Pancreatic progenitor cells represent both a potential source of transplantable islets for the treatment of diabetes and a valuable instrument for the investigation of the tumorigenesis of pancreatic carcinoma. It has been reported that pancreatic ductal cells of adults have the characteristics of pancreatic progenitors, but whether these cells can generate endocrine cells requires verification. Here, the differentiation of daughter cells of CD24(-) pancreatic ductal cells into insulin-secreting cells in vitro is reported. Crude pancreatic ductal cells were first obtained from adult mice by gradient centrifugation, and then the CD24(-) cells were isolated with a fluorescence-activated cell sorter. The isolated cells were cultured in serum-containing medium at clonal density to form epithelial colonies (ECs). The ECs were then stimulated with basic fibroblast growth factor (bFGF). After 72 h, insulin-secreting cells were observed in the ECs. These results indicate that the daughter cells of CD24(-) pancreatic ductal cells can differentiate into insulin-secreting cells in vitro when stimulated with exogenous bFGF. Therefore, CD24(-) pancreatic ductal cells have the potential to be pancreatic progenitor cells.