Germ line cell-derived pluripotent stem cells (GPSCs) are similar to embryonic stem (ES) cells in that they can proliferate intensively and differentiate into a variety of cell types. Previous studies have revealed some inherent differences in gene expression between undifferentiated mouse ES cells and GPSCs. Our aims were to generate functional hepatocytes from mouse GPSCs in vitro and to investigate whether the differences in gene expression may impact on the hepatocyte differentiation capacity of the GPSCs compared with ES cells. Mouse GPSCs and ES cells were induced to differentiate into hepatocytes through embryoid body formation, with very high efficiency. These hepatocytes were characterized at cellular, molecular, and functional levels. The GPSC-derived hepatocytes expressed hepatic markers and were metabolically active as shown by albumin and haptoglobin secretion, urea synthesis, glycogen storage, and indocyanine green uptake. We also performed an unprecedented DNA microarray analysis comparing different stages of hepatocyte differentiation. Gene expression profiling demonstrated a strong similarity between GPSC and ES cells at different stages of induced hepatic differentiation. Moreover, Pearson correlation analysis of the microarray datasets suggested that, at late hepatic differentiation stages, the in vitro-derived cells were closer to fetal mouse primary hepatocytes than to those obtained from neonates. We have shown for the first time that adult GPSCs can be induced to differentiate into functional hepatocytes in vitro. These GPSC-derived hepatocytes offer great potential for cell replacement therapy for a wide variety of liver diseases.