Transcription factors, such as PDX-1, that normally mediate pancreatic development are capable of inducing hepatic progenitor cells to differentiate into cells with pancreatic islet characteristics. We hypothesized that simultaneous expression of multiple transcription factors involved in islet development might enhance the differentiation of hepatic progenitor cells. Bi- or tri-cistronic constructs were generated in hybrid adenovirus/adeno-associated virus (Ad/AAV) vectors containing neurogenin 3 (NGN3), BETA2 (NeuroD), and RIPE3b1 (MafA), each of which plays a role in islet cell differentiation. These vectors efficiently express multiple transcription factors and stimulate insulin promoter activity in a combinatorial manner. When these multi-cistronic constructs were administered in vivo, they induce hepatic expression of islet-specific markers, including PDX-1, insulin, glucagon, somatostatin, and islet-amyloid peptide. Administration of the Ad/AAV hybrid vectors to streptozotocin-induced diabetic mice reversed hyperglycemia, consistent the differentiation of functional hepatic insulin-secreting cells. These results indicate that Ad/AAV hybrid vectors can be used to administer combinations of factors that induce islet cell differentiation in hepatic progenitor cells.