Introduction: Type I diabetes mellitus results from the autoimmune destruction of the β cells in pancreatic islets. Currently, extensive research is being conducted on the generation of insulin-producing cells (IPCs) from stem cells. P19 embryonal carcinoma cells are multipotent and can differentiate into cell types of all three germ layers. In this study, the differentiation of P19 cells into IPCs by using mouse pancreas extract (MPE) was investigated. Materials and Methods: Embryoid bodies (EBs) obtained from P19 cells were cultured in medium containing 3% fetal bovine serum, supplemented by concentration of 50, 100, 200,300 μg/mL MPE for 7-14 days. Dithizone (DTZ) staining was used to detect IPCs derived from EBs in vitro. Mouse monoclonal insulin-proinsulin and monoclonal insulin receptor beta antibodies were used for immunoflourescence. Insulin content from the cells and insulin secreted by differentiated cells in response to concentrations of 5.5 and 25 mM glucose were measured using ELISA kits. Results: DTZ-positive cells showed purple-red clusters. immunoflourescence indicated expression of Beta cell markers (insulin-proinsulin and insulin receptor beta) in these cells. Increasing glucose concentration, caused more insulin to be secreted by differentiated cells. Conclusions: P19 cells can in the presence of pancreas extract differentiate to cell producing and secreting insulin cells. Differentiated cells can increase insulin secretion in response to increasing glucose medium.