Activin A, which is a signaling molecule similar to Nodal, rapidly promotes endoderm induction of both embryonic stem (ES) cells and MSCs. Protocols for hepatic induction exhibit differences in efficiency and reproducibility depending on the specific methods or sources of MSCs. We characterized the effects of Activin A concentration on induction efficiency during hepatic differentiation of MSCs. Human MSCs (hMSCs) were differentiated into a hepatic lineage via a three-step protocol. Cells were first cultured in fetal bovine serum-free MSCs conditioned medium supplemented with Activin A (20, 50, or 100 ng/mL) for 3 days followed by treatment with additional agents. RT-PCR analysis, immunocytochemistry assays, periodic acid and Schiff's solution staining, and ELISAs were performed to confirm hepatic induction of hMSCs. Expression of genes related to the primitive foregut endoderm was observed in cells treated with higher concentration of Activin A. Gene expression related to functional primitive hepatocytes was observed in the early phases of hepatic differentiation. During the early period of the differentiation protocol, greater albumin secretion was observed when cells were treated with higher concentrations of Activin A. Thus, Activin A concentration affects the rate of endoderm induction of hMSCs, and at higher concentrations in vitro.