The low in vivo survival rate of scaffold-seeded cells is still a challenge in stem cell-based bone regeneration. This study seeks to use a silk hydrogel to deliver more stem cells into a bone defect area and prolong the viability of these cells after implantation. Rat bone marrow stem cells were mingled with silk hydrogels at the concentrations of 1.0 × 105/mL, 1.0 × 106/mL and 1.0 × 107/mL before gelation, added dropwise to a silk scaffold and applied to a rat calvarial defect. A cell tracing experiment was included to observe the preservation of cell viability and function. The results show that the hydrogel with 1.0 × 107/mL stem cells exhibited the best osteogenic effect both in vitro and in vivo. The cell-tracing experiment shows that cells in the 1.0 × 107 group still survive and actively participate in new bone formation 8 weeks after implantation. The strategy of pre-mingling stem cells with the hydrogel had the effect of delivering more stem cells for bone engineering while preserving the viability and functions of these cells in vivo.