Human hepatocyte transplantation entered clinical practice more than 10 years ago aiming to treat inborn errors of liver metabolism, and also fulminant liver failure with favorable results. However, a major challenge of hepatocyte transplantation is the limited supply of donor organs to isolate good quality cells. Hepatocytes for transplantation are obtained from steatotic livers, or those undergoing a long cold ischemia time, and also from surgical leftovers. We recently reported a phase I-II clinical study where freshly isolated human fetal liver cells (FLCs) were transplanted to bridge patients with chronic liver disease to solid organ transplantation, with encouraging and safe clinical results. The functional characterization of FLCs was conducted through the establishment of primary cultures, and by using adult hepatocytes and fetal liver MSC-like precursors as positive and negative control cells, respectively.On the basis of morphological and functional evaluations, we distinguished two groups of FLCstermed early-gestation (from 16 to 19 week-gestation) and late-gestation FLCs (from 20 to 22 week-gestation). The early-gestation FLC cultures were populated by various clusters of proliferating cells, while contained few hepatocytes. Consequently, they displayed poor liver-specific functions. On the contrary, the late-gestation FLC cultures contained cells that did not divide in vitro, but were functionally competent and similar to mature hepatocytes. Cultured 22-week-gestation fetal hepatocytes in particular, secreted albumin, synthesized urea and showed cytochrome P450 activity, isoform CYP3A4, and glucose-6-phosphatase enzymes at levels comparable to that of their adult counterparts. Treatment with dexamethasone in combination with oncostatin M induced a further maturation of fetal hepatocytes, which acquired additional functions, i.e., the ability to store glycogen and perform uptake of the vital stain indocyanine green (ICG). We also observed that the hepatic functionality was strongly dependent on culture conditions, with a low density culture system leading to rapid loss of the hepatocytic phenotype. In two weeks, these cultures became populated by spindle-like cells that were expanded up to 35 passages, and displayed a predominant mesenchymal phenotype after three subcultivations (approximately 75% CD90⁺, CD105⁺, and CD73⁺). On the basis of these observations, we conclude that late second trimester human fetal hepatocytes might be a valid alternative to adult hepatocytes in liver cell-based therapies, thus overcoming the difficulty of obtaining functional cells from unused livers for transplantation. Moreover, the high frequency of cells with features of precursors isolated from FLC cultures might improve the drawbacks of obtaining intrahepatic stem/precursors for clinical purposes, due to the low frequencies of cell precursors within the organ. Since FLCs can be long-term cryopreserved in the presence of 10% DMSO, without significant loss of viability and functions (e.g., plating efficiency and albumin secretion evaluated up to 1 year storage in liquid nitrogen), our results also suggest a novel approach for FLC transplantation by introducing the use of thawed cells, which will ultimately increase the number of available cells for transplantation.