Abstract.  Objectives: Recent studies have suggested the potential of mesenchymal stem cells (MSCs) to differentiate into a hepatocyte‐like lineage. Here, we evaluate the efficacy of hepatocyte differentiation of MSCs by studying acquisition of hepatocyte‐like features together with alteration of the native mesenchymal phenotype. Material and methods: In vitro, we have investigated protein and mRNA level expression of hepatocyte and mesenchymal markers of mesenchymal‐derived hepatocyte‐like cells (MDHLCs) and we have evaluated their functionality using metabolic assays. In vivo, we investigated co‐expression of hepatocyte (albumin, α‐foetoprotein, cytokeratin 18) and mesenchymal (fibronectin, vimentin) markers after transplantation of MSCs or MDHLCs into severe combined immune deficiency mice. Results: We observed that while in vitro these cells acquired some phenotypic and functional features of mature hepatocytes, they partially preserved their mesenchymal phenotype. After intrasplenic transplantation, engrafted MSCs with isolated expression of fibronectin and α‐foetoprotein were observed. When these cells were injected into the liver, they expressed all analysed markers, confirming the chimaeric co‐expression observed in vitro. Conversely, liver‐engrafted MDHLCs conserved their hepatocyte‐lineage markers but lost their chimaeric phenotype. Conclusions: Hepatocyte differentiation of MSCs predominantly allows the acquisition of phenotypic hallmarks and provides chimaeric cells that maintain expression of initial lineage markers. However, advanced maturation to the hepatocyte‐like phenotype could be obtained in vivo by conditioning MSCs prior to transplantation or by infusing cells into the liver micro‐environment.