In the epithelium of the developing glandular stomach, neuroendocrine cells differentiate from common progenitors, but the mechanism of how these cells are specified remains to be determined. Here, we show that the basic helix-loop-helix (bHLH) gene, mammalian achaete-scute homologue 1 (Mash1), is highly expressed in the glandular stomach epithelium. In Mash1-null mice, almost all gastric neuroendocrine cells are missing, whereas development of non-neuroendocrine cells is not significantly affected. The bHLH gene Neurogenin3 (Ngn3), which is known to regulate formation of subsets of gastric neuroendocrine cells (gastrin-, glucagon- and somatostatin-producing cells), is expressed normally in the Mash1-null stomach. Thus, Ngn3 alone is not sufficient but Mash1 is additionally required for the differentiation of these neuroendocrine cells. Taken together, these results indicate that formation of gastrin-, glucagon- and somatostatin-producing cells depends on both Mash1 and Ngn3, while that of other neuroendocrine cells depends on Mash1 alone, suggesting that combinations of bHLH genes may contribute to cell type diversity.