This study investigates how the mechanical properties of the extracellular matrix (ECM) influence the fate of mesenchymal stem cells (MSCs) in three-dimensional (3D) environments. The research demonstrates that the rigidity of the 3D matrix significantly affects the commitment of MSCs to different lineages, with osteogenesis predominantly occurring in matrices with intermediate stiffness (11–30 kPa). Unlike previous two-dimensional (2D) studies, where cell fate was correlated with morphology, this study shows that matrix stiffness regulates integrin binding and the reorganization of adhesion ligands on the nanoscale, both of which are traction-dependent and correlate with osteogenic commitment. These findings suggest that cells interpret changes in the physical properties of adhesion substrates as changes in adhesion ligand presentation, and that cells themselves can be harnessed as tools to mechanically process materials into structures that feedback to manipulate their fate. The study used hydrogels made from alginate, agarose, and poly(ethylene glycol dimethacrylate) (PEGDM) to create 3D matrices with varying stiffness. MSCs were encapsulated in these matrices, and their fate was analyzed after one week. The results showed that osteogenic commitment occurred primarily in matrices with intermediate stiffness, while adipogenic commitment was more common in softer matrices. The study also confirmed that the relationship between matrix elasticity and stem cell fate was consistent across different hydrogel types. The study further explored the underlying mechanisms of how matrix stiffness influences MSC fate. It found that the number of integrin-RGD bonds formed by MSCs was regulated by both the density of available RGD and matrix stiffness. The number of bonds peaked at 22 kPa, correlating with osteogenic commitment. The study also demonstrated that cell traction forces are necessary for matrix mechanics to regulate integrin binding, as blocking traction forces with BDM eliminated the effect of matrix stiffness on integrin binding. The study also showed that the mechanical properties of the matrix can influence the spatial organization of RGD peptides on the nanoscale, leading to clustering of RGD near integrins. This process is mediated by cell traction forces and is crucial for the formation of integrin-RGD bonds. The study used Förster Resonance Energy Transfer (FRET) to measure the number of integrin-RGD bonds and found that the number of bonds was regulated by both the density of RGD and matrix stiffness. Finally, the study confirmed that the fate of MSCs in 3D culture is mediated by integrin-RGD bonds. Blocking RGD-integrin bond formation with antibodies significantly diminished osteogenesis and enhanced adipogenesis, confirming the role of these bonds in determining cell fate. The study highlights the importance of matrix mechanics in controlling stem cell fate and suggests that cells can be harnessed as tools to mechanically process materials into structures that feedback to manipulate their fate. The findings have implications for the design of materialsThis study investigates how the mechanical properties of the extracellular matrix (ECM) influence the fate of mesenchymal stem cells (MSCs) in three-dimensional (3D) environments. The research demonstrates that the rigidity of the 3D matrix significantly affects the commitment of MSCs to different lineages, with osteogenesis predominantly occurring in matrices with intermediate stiffness (11–30 kPa). Unlike previous two-dimensional (2D) studies, where cell fate was correlated with morphology, this study shows that matrix stiffness regulates integrin binding and the reorganization of adhesion ligands on the nanoscale, both of which are traction-dependent and correlate with osteogenic commitment. These findings suggest that cells interpret changes in the physical properties of adhesion substrates as changes in adhesion ligand presentation, and that cells themselves can be harnessed as tools to mechanically process materials into structures that feedback to manipulate their fate. The study used hydrogels made from alginate, agarose, and poly(ethylene glycol dimethacrylate) (PEGDM) to create 3D matrices with varying stiffness. MSCs were encapsulated in these matrices, and their fate was analyzed after one week. The results showed that osteogenic commitment occurred primarily in matrices with intermediate stiffness, while adipogenic commitment was more common in softer matrices. The study also confirmed that the relationship between matrix elasticity and stem cell fate was consistent across different hydrogel types. The study further explored the underlying mechanisms of how matrix stiffness influences MSC fate. It found that the number of integrin-RGD bonds formed by MSCs was regulated by both the density of available RGD and matrix stiffness. The number of bonds peaked at 22 kPa, correlating with osteogenic commitment. The study also demonstrated that cell traction forces are necessary for matrix mechanics to regulate integrin binding, as blocking traction forces with BDM eliminated the effect of matrix stiffness on integrin binding. The study also showed that the mechanical properties of the matrix can influence the spatial organization of RGD peptides on the nanoscale, leading to clustering of RGD near integrins. This process is mediated by cell traction forces and is crucial for the formation of integrin-RGD bonds. The study used Förster Resonance Energy Transfer (FRET) to measure the number of integrin-RGD bonds and found that the number of bonds was regulated by both the density of RGD and matrix stiffness. Finally, the study confirmed that the fate of MSCs in 3D culture is mediated by integrin-RGD bonds. Blocking RGD-integrin bond formation with antibodies significantly diminished osteogenesis and enhanced adipogenesis, confirming the role of these bonds in determining cell fate. The study highlights the importance of matrix mechanics in controlling stem cell fate and suggests that cells can be harnessed as tools to mechanically process materials into structures that feedback to manipulate their fate. The findings have implications for the design of materials