Objective: Derivation, proliferation, and pluripotency of ESCs depend on their microenvironment, including the chemical and physical properties of the extracellular matrix (ECM) and the presence of growth factors. Surface modification of electrospun nanofibrous scaffolds have been studied to closely resemble natural ECMs and support in vitro and in vivo proliferation, pluripotency and differentiation of stem cells. Materials and Methods: In this study, we analyzed the maintenance of stemness and pluripotency of the mouse embryonic stem cell (mESC) following feederfree culture on collagen-grafted polyethersulfone (PESCOL) electrospun nanofibrous scaffold. Results: Our results showed that, the mESCs cultured for seven passages on PES-COL scaffolds had a typical undifferentiated morphology, enhanced proliferation, stable diploid normal karyotype, and continued expression of stemness and pluripotencyassociated markers, Oct-4, Nanog, SSEA-1, and Alkaline phosphatase (ALP) in comparison with PES scaffolds and gelatincoated plate. Moreover, these cells retained their in vitro and in vivo pluripotency. Our results indicated the enhanced infiltration and teratoma formation of mESCs in PES-COL. Conclusion: Here we demonstrate a successful feederfree culture system in which mESCs can be maintained an undifferentiated state and thereby eliminating the need of feeder layers for in vitro culture of embryonic stem cells. Furthermore, collagen-grafted nanofibrous scaffold could be a good candidate for feeder-free culture of human embryonic stem cells and induced pluripotent stem cells for clinical application.