Realization of clinical potential of human pluripotent stem cells (hPSCs) in bone regenerative medicine requires development of simple and safe biomaterials for expansion of hPSCs followed by directing their lineage commitment to osteoblasts. In the present study, a chemically defined peptide-decorated polycaprolactone (PCL) nano fibrous microenvironment was prepared through electrospinning technology and subsequent conjugation with vitronectin peptide to promote the culture and osteogenic potential of hPSCs in vitro. The results indicated that hPSCs successfully proliferated and maintained their pluripotency on the biointerface of peptide-conjugated nanofibers without Matrigel under defined conditions. Moreover, the prepared niche exhibited an appealing ability in promoting directed differentiation of hPSCs to osteoblastic phenotype without embryoid body formation step, determined from the cell morphological alteration, alkaline phosphate activity, and osteogenesis-related gene expression, as well as protein production. Such well-defined, xeno-free, and safe nanofiber scaffolds that allow the survival and facilitate osteo-differentiation of hPSCs provide a novel platform for hPSCs differentiation via cell-nanofiber interplay, and possess great value in accelerating the translational perspectives of hPSCs in bone tissue engineering.