The research on Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) from the umbilical cord suggests promising therapeutic use for hepatocyte replacement therapy. One of the highly conserved members of the nuclear receptor superfamily in the liver is hepatocyte nuclear factor-4α (HNF4α), involved in hepatocyte differentiation. The objectives of this study were to determine the effects of two- and three-dimensional (2D and 3D) cultures of WJ-MSCs on hepatocyte differentiation. MSCs were isolated from human Wharton's jelly, characterized by flow cytometry, and differentiated toward osteogenic and adipogenic lineage. WJ-MSCs were cultured in 2D collagen films and 3D collagen scaffolds in the presence of hepatogenic media with or without pre-treatment with fibroblast growth factor-4 (FGF4) for 21 days. The expression of HNF4α was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). According to flow cytometry data, the cells isolated from Wharton's jelly were shown to express MSC markers. HNF4α expression analysis revealed that pre-exposing the cells with FGF4 was more effective in hepatocyte differentiation. 3D cultures also improve the expression of HNF4α compared with 2D culture system. In conclusion, the combination of FGF4 and 3D culture improved hepatocyte differentiation. It seems 3D interaction of the cells improved the hepatogenesis.