Objective：To investigate whether adding different inducible factors：phosphatidylinositol 3-kinase（PI3K） inhibitor LY294002 and long-acting glucagon-like peptide-1（GLP-1） mimetic Exendin-4 could efficiently induce human embryonic stem cells（HESc） to differentiate into mature insulin-producing cells. Methods：A five-stage culturing strategy was used in this study. Human embryonic stem cells were amplified in vitro for 6－7 d at stage 1. Then，the HESc were cultured in the condition of suspension culture for forming embryoid bodies（EBs）（stage 2）. The nesting positive cells were selected in condition of ITSF medium（stage 3） and amplified in N2 and B27 medium（stage 4）. Finally，LY294002 was taken as experimental（group LY） and Exendin-4 was taken as control group（group Ex），which was utilized to induce the nesting positive cells into mature insulin-producing cells. The morphology and char－acteristics of each stage were observed under the microscope，and the specific markers were identified by immunofluorescence staining. Results：The positive rates of C-peptide staining（85.7±2.1） % vs. （74.5±2.7）%，P=0.005；somatostatin staining（75.4±3.7）% vs. （31.3±4.7）%，P=0.000；glucagon staining（38.1±3.4） % vs. （27.2±2.4）%，P=0.018；insulin/c-peptide（87.5±2.4）% vs. （76.7±3.1）%，P=0.013 and insulin/somatostatin co-staining（21.5±2.2）% vs. （3.5±0.9）%，P=0.000 in group Ex were significantly higher than those of group LY. Conclusion：LY294002 could induce HESc to differentiate into more mature insulin-produc－ing cells in serum-free culture medium.