Human embryonic stem cells can be differentiated into insulin-secreting cells by emulating in vitro the key processes that occur during embryonic development. However, the resulting cells are generally immature; thus, further research must be performed to identify the necessary factors to complete the differentiation. To this end, we cultured mouse embryonic stem cells with pancreatic bud-conditioned medium, based on a recent publication. Unlike in humans, mouse cells present two types of insulin, which can be used to identify different cell lineages. As a result, the cell product presented a neuroectodermal genetic expression pattern, with no expression of any definitive endodermal marker analyzed. Also, nonglucose-dependent insulin release was detected. Altogether, this previously published protocol results in neuroectoderm, and not definitive endoderm, derived insulin-positive cells. This further confirms the difficulty of obtaining true cell types of this germ layer. Finally, we identified a 16-kDa protein band that was present in pancreatic bud-conditioned medium. Sequencing this band revealed the presence of Reg proteins. The role of pancreatic bud-conditioned medium remains to be tested in definitive endoderm committed cells.