The differentiation of insulin-producing β-cells has been investigated in great detail; however, little is known about the factors that delineate the second-most abundant endocrine lineage, the glucagon-producing α-cell. Here we utilize a novel YAC-based Foxa3Cre transgene to delete the winged helix transcription factor Foxa2 (formerly HNF-3β) in the pancreatic primordium during midgestation. The resulting Foxa2^loxP/loxP; Foxa3Cre mice are severely hypoglycemic and die within the first week of life. Mutant mice are hypoglucagonemic secondary to a 90% reduction of glucagon expression. While the number of mature glucagon-positive α-cells is dramatically reduced, specification of α-cell progenitors is not affected by Foxa2 deficiency. By marker gene analysis, we show that the expression of the α-cell transcription factors Arx, Pax6, and Brn4 does not require Foxa2 in the transcriptional hierarchy governing α-cell differentiation.