Objective To compare the efficiencies of two methods (serum and non-serum) in inducing mouse embryonic stem cell differentiation into definitive endoderm cells. Methods The serum and non-serum methods were used to induce differentiation of mouse embryonic stem cells into definitive endoderm cells. Fluorescence activated cell sorter (FACS) was used to analyze the inducing time and efficiency of definitive endoderm using their surface protein marks (Cxcr4, c-Kit and E-cadherin). Meanwhile, RT-PCR was used to analyze the gene profile of definitive endoderm induced by the two methods. Real-time PCR was used to analyze the gene expression in definitive endoderm during the induction course in the non-serum group. The Cxcr4 and c-Kit double positive definitive endoderm cells were sorted by flow cytometry and gene profile was characterized by RT-PCR. Results Definitive endoderm cells were induced from mouse embryonic stem cells by both serum and non-serum methods. However, the efficiency of non-serum group (74.19%) was higher than that in the serum group, and the induction outcome reached a climax at the 4^th day of induction. Conclusion We have established a highly efficient method to induce differentiation of mouse embryonic stem cells into definitive endoderm, which lays a foundation for further differentiation into liver and pancreatic cells.