Recently, the expression of the peptide hormone ghrelin was detected in alpha-cells of the islets of Langerhans as well as in epsilon-cells, a newly discovered endocrine cell type, but it remains unclear how the latter is related in lineage to the four classical islet cell types, alpha-, beta-, delta-, and PP-cells. Here, we provide further evidence that ghrelin is predominantly produced in the alpha-cells of mouse islets but also in single hormone ghrelin-secreting epsilon-cells. We additionally demonstrate that pancreatic epsilon-cells derive from Neurogenin3-expressing precursor cells and their genesis depends on Neurogenin3 activity. Furthermore, our data indicate that the number of ghrelin-producing cells is differentially regulated during pancreas morphogenesis by the homeodomain-containing transcription factors Arx, Pax4, and Pax6. Arx mutants lack ghrelin+ glucagon+ alpha-cells whereas Pax4 mutants develop an excess of these cells. Importantly, the ghrelin+ glucagon- epsilon-cell population is not affected following Arx or Pax4 disruption. In contrast, the loss of Pax6 provokes an unexpected increase of the ghrelin+ glucagon- epsilon-cell number which is not due to increased proliferation. Thus, we demonstrate that the development of ghrelin-producing cells is differentially dependent on Neurogenin3 in different domains of the gastrointestinal tract and that, in the endocrine pancreas, epsilon-cell genesis does not require Arx or Pax4 activities but is antagonized by Pax6.