The mature differentiation of ES cells into hepatocytes and its differentiation ratio were studied in vitro. First, the BALB/c mouse ES cells were cultured for 5 days to develop into EBs in EBs culture medium. Then, the cell growth factors such as transform growth factor (TGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) were added into the medium to induce the differentiation of EBs cells into hepatocytes. During the culture medium, the reverse transcriptase-polymerase chain reaction (RT-PCR), immunocytochemistry (ICC) and radioimmunoassay (RIA) were used to determine the expression of hepatic genes and proteins, such as α-fetoprotein (AFP)，albumin (ALB)，glucose-6-phosphatase (G6P)，tyrosineaminotransferase (TAT)，cytokeratin 8 (CK8), cytokeratin 18 (CK18) and urea, to analyse the hepatic differentiation. At last, the hepatic differentiation ratio was also determined by counting the ICC-positive cells. In results, the mRNA of AFP, ALB, G6P and TAT didnt respectively expressed till at day3, day9, day11 and day13. AFP, ALB and urea in culture medium were not detected till at day8, day11 and day12 with a concentration of (3.4±0.6) μg/L, (0.21±0.04) mg/L and (8.3±1.4) μmol/L. Hepatic proteins such as AFP, ALB, CK8, CK18 didnt respectively expressed in cytoplasm till at day7, day9, day9, day11 and ICC-positive cells had morphological structures consistent with mouse primary hepatocytes. Hepatic differentiation ratio was determined at day19 with ratio of 32%. In conclusions, hepatocytes could be obtained in ES cells differentiation system and get matured with differentiation ratio of 32% when growth factors such as TGF, bFGF and HGF added into culture medium. This may produce a new resource of hepatocytes in liver engineering and hepatocytes transplantation for treating hepatic failure.