Peripheral blood mesenchymal stem cells (PBMSCs) may be easily harvested from patients, permitting autologous grafts for bone tissue engineering in the future. However, the PBMSC's capabilities of survival, osteogenesis and production of new bone matrix in the defect area are still unclear. Herein, PBMSCs were seeded into a nanofiber scaffold of self-assembling peptide (SAP) and cultured in osteogenic medium. The results indicated SAP can serve as a promising scaffold for PBMSCs survival and osteogenic differentiation in 3D conditions. Furthermore, the SAP seeded with the induced PBMSCs was splinted by two membranes of poly(lactic)-glycolic acid (PLGA) to fabricate a composited scaffold which was then used to repair a critical-size calvarial bone defect model in rat. Twelve weeks later the defect healing and mineralization were assessed by H&E staining and microcomputerized tomography (micro-CT). The osteogenesis and new bone formation of grafted cells in the scaffold were evaluated by immunohistochemistry. To our knowledge this is the first report with solid evidence demonstrating PBMSCs can survive in the bone defect area and directly contribute to new bone formation. Moreover, the present data also indicated the tissue engineering with PBMSCs/SAP/PLGA scaffold can serve as a novel prospective strategy for healing large size cranial defects.