Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. Investigating the roles of the co-culture of mouse embryonic stem cells (mESCs) with pancreatic islets (PL) and liver stromal cells (LSCs) in the differentiation of definitive endoderm (DE) cells was the purpose of this study. Here by using PL derived from adult mouse and LSCs derived from mouse fetal liver, we are trying to introduce a new protocol which is devoid of growth factors. We calculated mESCs indirectly for 7 days and then we analyzed the resulting cells regarding DE genes and protein expression using qRT-PCR and immunocytochemistry. It was proved that mESCs can differentiate into DE cells and also co-culture system with PL provides a proper environment for differentiation of mESCs. However co-culture system with LSCs isolated from mouse fetal liver, are not able to increase DE differentiation. As the first report we showed that a PL induced microenvironment can improve DE differentiation. More modifications of the PL microenvironment may present another approach to providing DE cells for differentiation into hepatocyte and beta cells. By applying these results, production of DE from stem cells facilitates in vitro.