The developmental potential of human embryonic stem cells (hESCs) holds great promise to provide a source of human hepatocytes for use in drug discovery, toxicology, hepatitis research, and extracorporeal bioartificial liver support. There are, however, limitations to induce fully functional hepatocytes on conventional two-dimensional (2D) static culture. It had been shown that dynamic three-dimensional (3D) perfusion culture is superior to induce maturation in fetal hepatocytes and prolong hepatic functions of primary adult hepatocytes. We investigated the potential of using a four-compartment 3D perfusion culture to induce hepatic differentiation in hESC. Undifferentiated hESC were inoculated into hollow fiber-based 3D perfusion bioreactors with integral oxygenation. Hepatic differentiation was induced with a multistep growth factor cocktail protocol. Parallel controls were operated under equal perfusion conditions without the growth factor supplementations to allow for spontaneous differentiation, as well as in conventional 2D static conditions using growth factors. Metabolism, hepatocyte-specific gene expression, protein expression, and hepatic function were evaluated after 20 days. Significantly upregulated hepatic gene expression was observed in the hepatic differentiation 3D culture group. Ammonia metabolism activity and albumin production was observed in the 3D directed differentiation culture. Drug-induced cytochrome P450 gene expression was increased with rifampicin induction. Using flow cytometry analysis the mature hepatocyte marker asialoglycoprotein receptor was found on up to 30% of the cells in the 3D system with directed hepatic differentiation. Histological and immunohistochemical analysis revealed structural formation of hepatic and biliary marker-positive cells. In contrast to 2D culture, the 3D perfusion culture induced more functional maturation in hESC-derived hepatic cells. 3D perfusion bioreactor technologies may be useful for further studies on generating hESC-derived hepatic cells.