Understanding the mechanisms that regulate hepatic epithelial cell differentiation are thought to be essential for the creation of efficient, programmed hepatic differentiation protocols from pluripotent stem cells. We here demonstrate that differences in response of pluripotent stem cells to cytokine-mediated lineage specification and differentiation between species will need to be taken into account, when inducing hepatic differentiation from ESC. During development of the mammalian liver, posterior epiblast cells from the blastocyst at first undergo a process called gastrulation, which results in the formation of mesendoderm, followed by DE and mesoderm. DE gives rise to the foregut, midgut and hindgut endoderm. The liver develops from the foregut endoderm, in response to factors secreted by the adjacent cardiac mesoderm and the septum transversum EX 527 mesenchyme. The foregut endoderm forms the liver bud, which contains bipotential hepatoblasts. Proliferation of the newly specified early hepatoblasts can be increased by other FGF’s. Induction of DE from mESC was initially described by embryoid body formation either alone or combined with growth factors mimicking development. More recently many groups have used monolayer cultures to induce DE and subsequently hepatic endoderm in mESC. These studies have demonstrated the importance of initial cell density, presence of serum, and the presence of Activin-A, for PS/DE induction. In addition, DE commitment from mESC and hESC in monolayer cultures appears to be enhanced by an inhibitor of GSK-3b or by Wnt3a conditioned medium. We described a protocol that supports directed differentiation of hESC in monolayer culture supplemented with 2% FCS to hepatocyte-like cells by sequential induction of PS/DE by ActivinA and Wnt3a, definitive and hepatic endoderm by BMP4 and FGF’s, and hepatocyte-like cells with HGF and Follistatin. Using the same hepatic differentiation protocol, but extending step 4 to 28 days, we here demonstrate that mESC cells from 129 and C57Bl/6 mice can be differentiated towards functional hepatocyte-like cells. Genes, characteristic for PS/DE were maximally and transiently expressed in response to Activin-A and Wnt3a on day 6, followed by formation of hepatic endoderm and finally gradual hepatic maturation. Hepatic maturation was similar in both mESC lines. The results of the functional assays were comparable with the functional results obtained when applying the protocol to human ESC.