This study reports a materials approach to tune the stress relaxation rate of hydrogels for 3D cell culture, independent of the hydrogel's initial elastic modulus, cell-adhesion-ligand density, and degradation. The research demonstrates that hydrogels with tunable stress relaxation can regulate stem cell fate and activity. Hydrogels composed of crosslinked polymer networks, such as polyethylene glycol (PEG), alginate, and hyaluronic acid, covalently coupled to integrin-binding ligands like RGD, are used for 3D cell culture or as cell-laden implants to promote tissue regeneration. These hydrogels offer independent control over physical and chemical properties, including matrix elasticity, ligand density, and porosity, and provide a homogeneous microenvironment. The study shows that hydrogels with faster stress relaxation enhance cell spreading, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs). MSCs cultured in rapidly relaxing hydrogels with an initial elastic modulus of 17 kPa form a mineralized, collagen-1-rich matrix similar to bone. The effects of stress relaxation are mediated by adhesion-ligand binding, actomyosin contractility, and mechanical clustering of adhesion ligands. The findings highlight stress relaxation as a key characteristic of cell-ECM interactions and an important design parameter for biomaterials in cell culture. The study investigates the impact of stress relaxation on cell behavior in 3D culture. It shows that hydrogels with faster stress relaxation promote cell spreading and proliferation, and enhance osteogenic differentiation of MSCs. The results indicate that stress relaxation influences MSC fate and activity through integrin-based adhesions, RGD ligand clustering, actomyosin contractility, and nuclear localization of YAP. The study also demonstrates that stress relaxation enables bone-forming activity in osteogenically differentiated stem cells. The research highlights the importance of considering matrix stress relaxation as a fundamental signal in understanding cell-ECM interactions and mechanotransduction. The findings suggest that stress relaxation can be used as a design parameter in tissue engineering to regulate cell proliferation and promote bone regeneration. The study also shows that the ability of cells to mechanically remodel their matrix is essential for cell-ECM interactions. The results indicate that stress relaxation is important for cells to respond to mechanical cues of the ECM. The study provides insights into the mechanisms underlying the effects of stress relaxation on cell behavior and MSC differentiation. The findings have implications for the development of biomaterials for tissue engineering and regenerative medicine.This study reports a materials approach to tune the stress relaxation rate of hydrogels for 3D cell culture, independent of the hydrogel's initial elastic modulus, cell-adhesion-ligand density, and degradation. The research demonstrates that hydrogels with tunable stress relaxation can regulate stem cell fate and activity. Hydrogels composed of crosslinked polymer networks, such as polyethylene glycol (PEG), alginate, and hyaluronic acid, covalently coupled to integrin-binding ligands like RGD, are used for 3D cell culture or as cell-laden implants to promote tissue regeneration. These hydrogels offer independent control over physical and chemical properties, including matrix elasticity, ligand density, and porosity, and provide a homogeneous microenvironment. The study shows that hydrogels with faster stress relaxation enhance cell spreading, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs). MSCs cultured in rapidly relaxing hydrogels with an initial elastic modulus of 17 kPa form a mineralized, collagen-1-rich matrix similar to bone. The effects of stress relaxation are mediated by adhesion-ligand binding, actomyosin contractility, and mechanical clustering of adhesion ligands. The findings highlight stress relaxation as a key characteristic of cell-ECM interactions and an important design parameter for biomaterials in cell culture. The study investigates the impact of stress relaxation on cell behavior in 3D culture. It shows that hydrogels with faster stress relaxation promote cell spreading and proliferation, and enhance osteogenic differentiation of MSCs. The results indicate that stress relaxation influences MSC fate and activity through integrin-based adhesions, RGD ligand clustering, actomyosin contractility, and nuclear localization of YAP. The study also demonstrates that stress relaxation enables bone-forming activity in osteogenically differentiated stem cells. The research highlights the importance of considering matrix stress relaxation as a fundamental signal in understanding cell-ECM interactions and mechanotransduction. The findings suggest that stress relaxation can be used as a design parameter in tissue engineering to regulate cell proliferation and promote bone regeneration. The study also shows that the ability of cells to mechanically remodel their matrix is essential for cell-ECM interactions. The results indicate that stress relaxation is important for cells to respond to mechanical cues of the ECM. The study provides insights into the mechanisms underlying the effects of stress relaxation on cell behavior and MSC differentiation. The findings have implications for the development of biomaterials for tissue engineering and regenerative medicine.