Objective To define the best culture conditions of ES-R1 and explore the possibility and feasibility of differentiation of ES-R1 to IPCs in vitro with various of factors. Methods ESCs were cultured on the mitomycin Ctreated MEF feeder layer cells. The culture medium of ES-R1 was supplemented with 10μg/L LIF. ESCs were transferred into the ultralow adherent dishes in the absence of LIF and feeder layers for the formation of EBs. On day 4 of EBs formation, EBs were transferred to collagen-Coated dishes and serumfree ITSA medium, pancreatic proliferation medium and pancreatic differentiation medium were added step by step, each for 6 days. During the differentiation from ESCs to IPCs, morphological methods, immunofluoresence staining, alkaline phosphatase staining, dithizong staining and insulin release test were used. Results Undifferentiated state was maintained by feeder cells and LIF. ESCs could form nested colonies with clear edge and smooth surface. Upon withdrawal of LIF and feeder layer, ESCs spontaneously formed into EBs in suspension. At the end of induction of differentiation, the three dimensional IPCs were formed. The induced IPCs were found to be stained crimson red by DTZ, insulin and glucagon immunohistochemistry staining positive, insulin release test positive.Conclusion Combination of LIF and MEF feeder layer culture may maintain the proliferation of ES cells without differentiation. Various factors used at different times, ES-R1 can differentiate into IPCs which possess ch