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Objective: To study the impact of ultraweb nano-fibrillar substrates on in vitro differentiationof mouse bone marrow-derived mesenchymal stem cells into hepatocyte-like cells.Materials and Methods: For a duration of up to 36 days, mouse bone marrow mesenchymalstem cells (MSCs) were cultured on an artificial basement membrane containingultraweb nanofiber (nano+) and plates (nano-) coated with Matrigel and used growth anddifferentiating factors for hepatogenic differentiation. Then, to study the impact of nanofiberson hepatocyte differentiation, mRNA expressions for liver-specific genes (CK7, AFP,FOXa2, ALB, CYP1a1, HNF4a) were analyzed using real-time PCR , and ultrastructuresof differentiated cells were evaluated using scanning and transmission electron microscope.Results: Both experimental groups expressed mRNA of liver-specific genes in a timedependent manner. However, mRNA expression trends of liver-specific genes, especiallythose of FOXa2 and HNF4a, were better in the nano+ culture as compared to the nanoculture(p<0.05). Furthermore, the ultrastructures of differentiated cells in the 36 day-oldnano+ culture were more mature as compared to those of the nano- group.Conclusion: The results suggest that topographical properties of the extracellular matricesproduced by ultraweb nanofibers are effective for the in vitro reproduction of moredifferentiated MSC-derived hepatocyte-like cells.