A basic question linked to differential patterns of gene expression is how cells reach different fates despite using the same DNA template. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome, including a role in establishing such tissue-specific patterns of expression. Recently we described TET1-mediated enrichment of 5hmC on the promoter region of the master regulator of hepatocyte identity, HNF4A, which precedes differentiation of liver adult progenitor cells in vitro. Here, we studied the genome-wide distribution of 5hmC at early in vitro differentiation of human hepatocyte-like cells. We found a global increase in 5hmC as well as a drop in 5-methylcytosine after one week of in vitro differentiation from bipotent progenitors, at a time when the liver transcript program is already established. 5hmC was overall higher at the bodies of overexpressed genes. Furthermore, by modifying the metabolic environment, an adenosine derivative prevents 5hmC enrichment and impairs the acquisition of hepatic identity markers. These results suggest that 5hmC could be a marker of cell identity, as well as a useful biomarker in conditions associated with cell de-differentiation such as liver malignancies.