Background <p>Chicken hepatocytes are a valuable resource for cell-based assays and investigating the underlying mechanisms of diseases.</p> Objective <p>In this study, we examined in vitro generation of chicken hepatocytes from three embryonic developmental stages.</p> Methods <p>Hepatocytes were isolated from 5-, 7-, and 10-day-old chicken embryos and cultured in DMEM/F12 + 10% FBS. After 3 days, we measured proliferation rate, the expression of hepatocyte-specific genes (AFP, ALP, FOXA2, CYP3A4, CXCR4, OCT4, NANOG, SOX17), enzyme activity (ALT, AST), albumin production, and urea secretion.</p> Findings <p>Morphological examination of individual hepatocytes exhibited a characteristic hexagonal structure with prominent nuclei and nucleoli. E10 embryos exhibiting a markedly higher proliferation rate in comparison to those from E5 and E7 stages (until 20 days). Whereas, approximately 50% of E5 and E7 hepatocytes showed reduced proliferation after three days. In suspension culture, hepatocytes formed spheroids or hepatospheres. The expression of hepatocyte-specific genes (AFP, ALP, FOXA2, and CYP3A4) was higher in E10 compared to E5 hepatocytes. The expression of stemness/early developmental markers (CXCR4, OCT4, NANOG, and SOX17) was significantly lower in E10 than E5 hepatocytes. E10 hepatocytes revealed significantly increased ALT and AST expression and urea secretion. While, Albumin production was significantly lower in E10 hepatocytes.</p> Conclusion <p>Our results invested that E10 is the optimal developmental stage for the derivation and proliferation of chicken hepatocytes in vitro.</p>