The liver, the largest gland in the body, performs over 500 essential functions and displays a remarkable capacity for regeneration. When compromised, this regenerative ability can lead to severe conditions such as liver cirrhosis and hepatocellular carcinoma. A potential solution to these liver conditions lies in vitro hepatocyte differentiation from pluripotent stem cells, including human embryonic stem cells and induced pluripotent stem cells. Our study explored the role of epigenetic modifications in this process, specifically DNA methylation and hydroxymethylation. These modifications are crucial in modulating gene expression and cellular phenotypes, which influence the differentiation and function of cells. The results demonstrated significant differential methylation in key regions associated with hepatocyte differentiation, implicating their potential importance in successful in vitro hepatocyte differentiation. Additionally, the utility of the Infinium Methylation EPIC BeadChip for high-throughput analysis was confirmed, revealing a broad spectrum of methylation and hydroxymethylation changes during hepatocyte differentiation. The findings suggest a previously underappreciated role of DNA methylation and hydroxymethylation in influencing hepatocyte differentiation, with potential implications for enhancing the efficiency and stability of in vitro hepatocyte generation. This understanding can inform the development of improved protocols, providing valuable platforms for studying liver diseases and developing novel therapeutics. Future studies will continue to dissect the intricate network of epigenetic regulations, opening new avenues for understanding and treating liver diseases.