Embryonic stem (ES) cells can replicate indefinitely and differentiate into all cell types, including hepatocytes. Research using primate ES cells is considered to be important for studies of potential cell therapies. Recently, we established cynomolgus monkey ES cells designated as CMK6. The CMK6 cell line is a useful tool for investigating the mechanism of differentiation in primate ES cells and developing cell therapies, because of its biological similarity to human ES cells. To examine whether cynomolgus monkey ES cells differentiate into hepatocytes, CMK6 cells were cultured with or without acidic fibroblast growth factor (aFGF). Evaluation of the hepatic differentiation was performed by analysis of the mRNA expression in early hepatic marker genes using the reverse transcriptase-polymerase chain reaction (RT-PCR). The protein expression of albumin (ALB) was also studied by immunocytochemistry. RT-PCR analyses revealed mRNA expressions of alpha-fetoprotein, transthyretin, and ALB in the presence of aFGF at 3 wk of differentiation, whereas no mRNA expression of these genes was detected in cells without aFGF. The protein expression of ALB in the presence of aFGF at 3 wk of differentiation was also confirmed by immunocytochemistry. However, tyrosine aminotransferase, which is a mature hepatic marker, was not detected in the presence or absence of aFGF at any stage of differentiation. These results suggested that aFGF successfully promoted in vitro differentiation of cynomolgus monkey ES cells to an early hepatic lineage.