To investigate whether insulin-producing cells obtained from ES cells via the nestin-positive cell-mediated method are of the pancreatic lineage, we established a pdx-1 knockout ES cell line and analyzed its differentiation into insulin-producing cells. As a result, pdx-1 knockout ES cell expressed insulin 2 gene at the final differentiated cells. Thus, our study demonstrated that pdx-1 is not essential for insulin gene expression, at least in cells differentiated from this population of nestin-expression enriched ES cells, and suggested that the insulin-producing cells derived from ES cells may be different from the pancreatic beta cells in terms of their lineage.