For long-term treatment of diabetes type 1, transplantation of insulin-producing beta cells may be a promising method, but the limited number of islets for transplantation requires the development of different approaches. In this study, we aimed to generate betalike insulin-producing cells. For this purpose, MafA, Pax4, and Ngn3 genes were transferred into pancreatic islet-derived mesenchymal stem cells, and the effect of their ectopic expressions on differentiation efficiency was examined. Stemness properties of pancreatic islet stem cells were characterized. The 3 genes were transfected by electroporation and expressed constitutively. The transfected cells were further stimulated to differentiate by using chemical induction. Pax4 expression had significant effects on differentiation into insulin-producing cells. Although it caused morphological alterations in cells, similar to epithelial cells, the insulin secretion levels remained lower than those of the cell line cotransfected with MafA and Pax4. Cotransfection of the 3 transcription factors did not further improve the beta-like cell generation. MafA and Pax4 ectopic expression resulted in improved differentiation efficiency into insulin-secreting cells. However, support of this differentiation process using additional chemical induction may suffice to overcome control by endogenous regulatory pathways.