In this study, we compared the efficiency of osteoblast differentiation media (ODM) containing three distinct reagent combinations in osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) in monolayer culture. In addition, we analyzed growth and differentiation of hBMSCs on silk scaffolds and examined the bone-forming activity of a nanofibrous silk scaffold in a tibia diaphysis defect model of a rat hind limb with intramedullary nailing. Although all three ODM increased alkaline phosphatase activity to a comparable extent, the ODM containing bone morphogenetic protein-2 (BMP-2) was found to be significantly less effective in promoting mineral deposition than the others. Growth of hBMSCs on sponge-form silk scaffolds was faster than on nanofibrous ones, while osteoblastic differentiation was apparent in the cells grown on either type of scaffold. By contrast, bone formation was observed only at the edge of the nanofibrous scaffold implanted in the tibia diaphysis defect, suggesting that use of the silk scaffold alone is not sufficient for the reconstitution of the long bone defect. Since silk scaffolds can support cell growth and differentiation in vitro, loading MSCs on scaffolds might be necessary to improve the bone-forming activity of the scaffold in the long bone defect model.