Abstract: Objective To establish a system in which mouse embryonic stem cells (mESCs) differentiated into insulin-secreting cells in vitro. Methods mESCs were cultured to form embryoid bodies(EBs). EBs were cultured in serum-free medium to obtain nestin-positive cells. The selected nestin-positive cells were expanded, then added different concentration of nicotinamide into the medium to induce the differentiation of nestin-positive cells. Immunochemistry staining and flow cytometry analysis were made on 15th day after induction. Results Flow cytometry analysis revealed that the percentage of nestin-positive cells were different from EBs whose diameters were different. The percentage of nestin-positive cells from EBs whose diameters about 100μm were higher than those of other diameters. Nestin-positive cells induced by nicotinamide could form islet-like cell clusters. Flow cytometry analysis revealed the percentages of insulin-positive cells were different after induced for 15 days by different concentration nicotinamide. Percentage of insulin-positive cell induced by 10mmol/L nicotinamide were higher than those induced by 0 mmol/L or 5 mmol/L nicotinamide(P<0.01). The induced cells could secrete insulin in responding to different glucose concentration. Conclusion More insulin-secreting cells could be obtained from the nestin-positive cells after treatment of 10 mmol/L nicotinamide for 15 days.