Multipotential stem cells can be selected from the bone marrow by plastic adhesion, expanded, and cultured. They are able to differentiate not only into multiple cell types, including cartilage, bone, adipose and fibrous tissues, and myelosupportive stroma, but also into mesodermal (endothelium), neuroectodermal, or endodermal (hepatocytes) lineages. Our goal was to characterize the multipotential capacities of human mesenchymal stem cells (hMSCs) and to evaluate their ability to differentiate into insulin-secreting cells in vitro. hMSCs were obtained from healthy donors, selected by plastic adhesion, and phenotyped by fluorescence-activated cell sorter and reverse transcription-polymerase chain reaction analysis before and after infection with adenoviruses coding for mouse IPF1, HLXB9, and FOXA2 transcription factors involved early in the endocrine developmental pathway. We found that native hMSCs have a pluripotent phenotype (OCT4 expression and high telomere length) and constitutively express NKX6-1 at a low level but lack all other transcription factors implicated in beta-cell differentiation. In all hMSCs, we detected mRNA of cytokeratin 18 and 19, epithelial markers present in pancreatic ductal cells, whereas proconvertase 1/3 mRNA expression was detected only in some hMSCs. Ectopic expression of IPF1, HLXB9, and FOXA2 with or without islet coculture or islet-conditioned medium results in insulin gene expression. In conclusion, our results demonstrated that in vitro human bone marrow stem cells are able to differentiate into insulin-expressing cells by a mechanism involving several transcription factors of the beta-cell developmental pathway when cultured in an appropriate microenvironment.