Many homeodomain transcription factors function in organogenesis and cell differentiation. The Nkx family illustrates these functions especially well, and the Nkx6 subfamily controls differentiation in the central nervous system and pancreas. Nkx6.3, a recent addition to this subfamily, overlaps Nkx6.1 and Nkx6.2 in expression in the hindbrain and stomach. Nkx6.3 transcripts localize in the epithelium of the most distal stomach region, the antrum and pylorus; expression in the adult intestine is lower and confined to the proximal duodenum. Nkx6.3(-)(/)(-) mice develop and grow normally, with a grossly intact stomach and duodenum. These mice show markedly reduced gastrin mRNA, many fewer gastrin-producing (G) cells in the stomach antrum, hypogastrinemia, and increased stomach luminal pH, with a corresponding increase in somatostatin mRNA levels and antral somatostatin-producing (D) cells. They express normal levels of other transcription factors required for gastric endocrine cell differentiation, Pdx1, Pax6, and Ngn3; conversely, Ngn3(-)(/)(-) mice, which also show reduced gastrin levels, express Nkx6.3 normally. These studies implicate Nkx6.3 as a selective regulator of G- and D-cell lineages, which are believed to derive from a common progenitor, and suggest that it operates in parallel with Ngn3.