A silk-based hydrogel system was explored to evaluate the effects of substrate stiffness and cyclic stretch on human mesenchymal stem cell (hMSCs) differentiation. The hydrogel fibers were highly tunable, elastic and supported the survival and growth of encapsulated hMSCs. The hMSCs in silk hydrogel fibers at 50 kPa and 150 kPa both transferred from a multipotent stem cell state to a pre-osteoblast/adipocyte state in static culture. With cyclic stretching, osteo-differentiation was enhanced, while adipo-differentiation was suppressed. A new method to generate a tunable, anisotropic 3D porous scaffold with branching networks to enhance vascularization in vitro and direct anastomosis in vivo was also developed. Branched channels were generated in the silk-based scaffolds with proper endothelialization. Silk fibers in the scaffold aligned with a specific direction and achieved anisotropic diffusion of molecules with different sizes. These new silk-based tissue engineered constructs can integrate physical and chemical cues for desired stem cell differentiation to improve tissue regeneration.