HNF-4α is a transcription factor of the nuclear hormone receptor family that is expressed in the hepatic diverticulum at the onset of liver development. Mouse embryos lacking HNF-4α fail to complete gastrulation due to dysfunction of the visceral endoderm. This early embryonic lethality has so far prevented any analyses of the contribution of HNF-4α toward liver development and hepatocyte differentiation. However, we have shown that complementation ofHNF-4 α ^−/−embryos with a tetraploid embryo-derived wild-type visceral endoderm rescues this early developmental arrest and allowsHNF-4 α ^−/−embryos to proceed normally through midgestation stages of development. Examination of these rescued embryos revealed that HNF-4α was dispensable for specification and early development of the liver. However,HNF-4α ^−/− fetal livers failed to express a large array of genes whose expression in differentiated hepatocytes is essential for a functional hepatic parenchyma, including genes encoding several apolipoproteins, metabolic proteins, and serum factors. In addition, we have demonstrated that HNF-4α is essential for expression of the transcription factors HNF-1α and PXR within the fetal liver. We therefore conclude that HNF-4α is both essential for hepatocyte differentiation during mammalian liver development and also crucial for metabolic regulation and liver function.