Previously, we reported a system to enrich mouse fetal hepatic progenitor cells (HPCs) by forming cell aggregates. In this study, we sorted two cell populations, CD49f⁺Thy1⁻CD45⁻ cells (CD49f‐postive cells) and CD49f^±Thy1⁺CD45⁻ cells (Thy1‐positive cells), from the cell aggregates using a flow cytometer. CD49f‐positive cells stained positive for endodermal specific markers such as α‐fetoprotein (AFP), albumin (ALB), and cytokeratin 19 (CK19), and are thus thought to be HPCs. However, Thy1‐positive cells were a morphologically heterogeneous population; reverse‐transcription polymerase chain reaction (RT‐PCR) and immunocytochemical analyses revealed the expression of mesenchymal cell markers such as α‐smooth muscle actin, desmin, and vimentin, but not of AFP, ALB, or CK19. Therefore, Thy1‐positive cells were thought to be of a mesenchymal lineage. When these two cell populations were co‐cultured, the CD49f‐positive colonies matured morphologically and stored a significant amount of glycogen. Furthermore, real‐time RT‐PCR demonstrated an increased expression of tyrosine amino transferase and tryptophan oxygenase mRNA, and transmission electron microscopy confirmed that co‐cultured cells produced mature hepatocytes. However, when CD49f‐positive cells were cultured alone or when the two populations were cultured separately, the CD49f‐positive cells did not mature. These results indicate that CD49f‐positive cells are primitive hepatic endodermal cells with the capacity to differentiate into hepatocytes, and that Thy1‐positive cells promote the maturation of CD49f‐positive cells by direct cell‐to‐cell contact. In conclusion, we were able to isolate CD49f‐positive primitive hepatic endodermal cells and Thy1‐positive mesenchymal cells and to demonstrate the requirement of cell‐to‐cell contact between these cell types for the maturation of the hepatic precursors. (HEPATOLOGY 2004;39:1362–1370.)