Elucidating the complex molecular regulatory mechanisms underlying hepatic differentiation at early stage contributes both to fully harness embryonic stem cells in liver regeneration, and to understand the differentiation-related liver diseases. Pluripotent embryonic stem cells can be induced into hepatocytes in vitro. mESC-D3, maintained in adherent monolayer culture condition, were induced to differentiate along hepatic lineage with addition of some factors to the medium at different time. The factors included FGF, HGF, OSM and so on. The differentiated cells showed a hepatocyte-like morphology, expressed hepatic marker genes and have also produced and stored glycogen by phase contrast and transmission electron microscopy, reverse transcription-polymerase chain reaction, immunocytochemistry, and periodic acid-Shiff staining respectively. Stem cell differentiation-related microarray was used to analyze the differential gene expression profiling during hepatic differentiation of mESC-D3 at early stage. Quantitative PCR was performed to verify the microarray data. Microarray analysis presented 48 genes expressed differentially (2 fold), including 20 genes up-regulated and 28 genes down-regulated. Further bioinformatics analysis showed the majority of these genes were extracellular matrix, intercellular junction and FGF, BMP, Notch and Wnt signaling pathways molecules, which suggests these alterations may be closely associated with the hepatic differentiation of embryonic stem cells at early stage.