Embryonic stem cells possess the ability to differentiate in vitro into a variety of cell lineages, including insulin-producing cells. Pancreatic beta-cells derive from foregut endoderm during embryonic development. However, previous reports using transgenic mice strongly indicate that insulin-positive cells may be generated also through the neuroectoderm pathway. To analyze this point, a culture system was performed in which only ectoderm committed cells were present. Based on published work, we achieved this by maintaining transfected clonal R1 mouse embryonic stem cells in monolayer in the absence of LIF. Contrary to differentiation protocols via embryoid body formation, monolayer cultured cells displayed ectodermal fates according to the marker gene expression pattern. Under these particular conditions, neomycin was added in order to select insulin-expressing cells. The cell lineage obtained expressed Pdx1, Pax6, Isl1, AChE, MBP, TH, and GS genes, confirming ectodermal commitment, even though some of these factors are also expressed in endoderm. In addition these cells displayed excitatory properties similar to astrocytes. Co-expression of insulin II and nestin was observed in monolayer culture and in the presence of specific conditioned media. No expression of early endodermal markers was detected along monolayer cultures. Altogether, these observations suggest that cells with ectoderm fates could participate in vitro in the derivation of insulin-producing cells. These results have implications for insulin gene regulation and hormone secretion in order to generate insulin-producing cells for replacement protocols in the treatment of diabetes.