Objective: Embryonic stem cells (ESCs) culture and its applications are limited by the use of mouse embryonic ﬁbroblasts (MEFs) as a feeder layer. Xenogeneic contaminants and variability in MEFs from batch-to-batch and laboratory-to-laboratory restricted the culture and transplantation of these cells. Thus, the development of feeder-free culture system is important to overcome this limitation. Materials and Methods: In this study we demonstrated a successful feeder-free culture system for undifferentiated mouse ESCs expansion using polyethersulfone (PES) nanofiber scaffold. Analyses of alkaline phosphatase activity, Oct-4 immunocytochemistry and the gene expression of pluripotency markers has been performed after feeder free culture of mESCs on the PES nanofiber scaffold. Results: The results of our culture system revealed that the characteristics of undifferentiated cells such as alkaline phosphatase activity, Oct-4 immunoreactivity and the gene expression of pluripotency markers (Oct-4 and Nanog) than the cells cultured on a conventional gelatin-coated surface. In addition the cultured cells retained the in vitro differentiation potential to the three embryonic germ layers. Conclusion: These results indicate that this modiﬁed culture surface may be a useful tool for obtaining enriched preparations of undifferentiated ESCs.