Differentiation of human embryonic stem cells (hESCs) to specific functional cell types can be achieved using methods that mimic in vivo embryonic developmental programs. Current protocols for generating hepatocytes from hESCs are hampered by inefficient differentiation procedures that lead to low yields and large cellular heterogeneity. We report here a robust and highly efficient process for the generation of highpurity (70%) hepatocyte cultures from hESCs that parallels sequential hepatic development in vivo. Highly enriched populations of definitive endoderm were generated from hESCs and then induced to differentiate along the hepatic lineage by the sequential addition of inducing factors implicated in physiological hepatogenesis. The differentiation process was largely