A strategy is presented for creating photodegradable poly(ethylene glycol)-based (PEG) hydrogels through rapid polymerization of cytocompatible macromers for remote manipulation of gel properties in situ. Post-gelation control of the gel properties allows for temporal changes, creation of arbitrarily shaped features, and on-demand release of pendant functionality. Channels photodegraded within a hydrogel containing encapsulated cells allow cell migration. Temporal variation of the biochemical gel composition is utilized to influence chondrogenic differentiation of encapsulated stem cells. Photodegradable gels that allow real-time manipulation of material properties or chemistry provide dynamic environments for studying material regulation of live cell function and may impact applications from drug delivery to tissue engineering. Hydrogels are hydrophilic polymers swollen by water that are insoluble due to physical or chemical crosslinks. They are used extensively as biomaterials for complex device fabrication, cell culture for tissue regeneration, and targeted drug release. Control of gel structure in space and time is required to understand the dynamic relationship between biomaterial properties and their influence on biological function. For example, progenitor cells are often expanded and differentiated in hydrogel microenvironments, and researchers have demonstrated how initial gel properties, including mechanics and chemical functionality, influence cellular fate. In regenerative medicine, the structure and composition of gels are regulated temporally through hydrolytic and enzymatic degradation mechanisms to promote cell secretory properties and encourage the development of tissue-like structures in vitro and in vivo. Hydrogel structure and functionality have evolved from the direct encapsulation of cells in simple homogeneous materials to those with highly regulated structures spanning multiple size scales. These hydrogel structures are further modified locally by cells with the synthetic incorporation of bioresponsive functionalities or externally by advanced patterning to create spatially varying functionalities. For example, the chemical patterning of a gel by the addition of a second, interpenetrating network or peptide tether has been demonstrated by diffusing chemical moieties into a gel and covalently linking these functionalities to the network by photocoupling or reaction with a photolytically uncaged reactive group. While these are important advances, such processes do not allow modulation of the gel chemistry in real-time or photodegradation of the gel structure. Few synthetic materials provide a cellular microenvironment in which physical or chemical cues are initially present and subsequently regulated on-demand. The authors have synthesized monomers capable of polymerizing in the presence of cells to produce photolytically degradable hydrogels whose physical or chemical properties are tunable temporally and spatially with light. The desired gel property for altering cell function or fabricating a device is thus externally-triggered and directed with irradiation by photolytic cleavage and removal of the macromolecules that comprise the gel. The photodegradable functionality, a nitrobenzyl ether-derived moiety, was selected based on its photolytic efficiency, susceptibility to two-photon photolysisA strategy is presented for creating photodegradable poly(ethylene glycol)-based (PEG) hydrogels through rapid polymerization of cytocompatible macromers for remote manipulation of gel properties in situ. Post-gelation control of the gel properties allows for temporal changes, creation of arbitrarily shaped features, and on-demand release of pendant functionality. Channels photodegraded within a hydrogel containing encapsulated cells allow cell migration. Temporal variation of the biochemical gel composition is utilized to influence chondrogenic differentiation of encapsulated stem cells. Photodegradable gels that allow real-time manipulation of material properties or chemistry provide dynamic environments for studying material regulation of live cell function and may impact applications from drug delivery to tissue engineering. Hydrogels are hydrophilic polymers swollen by water that are insoluble due to physical or chemical crosslinks. They are used extensively as biomaterials for complex device fabrication, cell culture for tissue regeneration, and targeted drug release. Control of gel structure in space and time is required to understand the dynamic relationship between biomaterial properties and their influence on biological function. For example, progenitor cells are often expanded and differentiated in hydrogel microenvironments, and researchers have demonstrated how initial gel properties, including mechanics and chemical functionality, influence cellular fate. In regenerative medicine, the structure and composition of gels are regulated temporally through hydrolytic and enzymatic degradation mechanisms to promote cell secretory properties and encourage the development of tissue-like structures in vitro and in vivo. Hydrogel structure and functionality have evolved from the direct encapsulation of cells in simple homogeneous materials to those with highly regulated structures spanning multiple size scales. These hydrogel structures are further modified locally by cells with the synthetic incorporation of bioresponsive functionalities or externally by advanced patterning to create spatially varying functionalities. For example, the chemical patterning of a gel by the addition of a second, interpenetrating network or peptide tether has been demonstrated by diffusing chemical moieties into a gel and covalently linking these functionalities to the network by photocoupling or reaction with a photolytically uncaged reactive group. While these are important advances, such processes do not allow modulation of the gel chemistry in real-time or photodegradation of the gel structure. Few synthetic materials provide a cellular microenvironment in which physical or chemical cues are initially present and subsequently regulated on-demand. The authors have synthesized monomers capable of polymerizing in the presence of cells to produce photolytically degradable hydrogels whose physical or chemical properties are tunable temporally and spatially with light. The desired gel property for altering cell function or fabricating a device is thus externally-triggered and directed with irradiation by photolytic cleavage and removal of the macromolecules that comprise the gel. The photodegradable functionality, a nitrobenzyl ether-derived moiety, was selected based on its photolytic efficiency, susceptibility to two-photon photolysis