Defects in pancreatic -cell function contribute to the development of type 2 diabetes, a polygenic disease that is characterized by insulin resistance and compromised insulin secretion. Hepatocyte nuclear factors (HNFs) -1␣, -3, -4␣, and Pdx-1 contribute in the complex transcriptional circuits within the pancreas that are involved in -cell development and function. In mice, a heterozygous mutation in Pdx-1 alone, but not Hnf-1␣ ؉/؊ , Hnf-3 ؉/؊ , or Hnf-4␣ ؉/؊ , causes impaired glucose-stimulated insulin secretion in mice. To investigate the possible functional relationships between these transcription factors on -cell activity in vivo, we generated mice with the following combined heterozygous mutations: Pdx-, and Hnf-3 ؉/؊ ͞Hnf-4␣ ؉/؊ . The greatest loss in function was in combined heterozygous null alleles of Pdx-1 and Hnf-1␣ (Pdx-1 ؉/؊ ͞Hnf-1␣ ؉/؊ ), or Pdx-1 and Hnf-3 (Pdx-1 ؉/؊ ͞Hnf-3 ؉/؊ ). Both double mutants develop progressively impaired glucose tolerance and acquire a compromised first-and second-phase insulin secretion profile in response to glucose compared with Pdx-1 ؉/؊ mice alone. The loss in -cell function in Pdx-1 ؉/؊ ͞Hnf-3 ؉/؊ mice was associated with decreased expression of Nkx-6.1, glucokinase (Gck), aldolase B (aldo-B), and insulin, whereas Nkx2.2, Nkx-6.1, Glut-2, Gck, aldo-B, the liver isoform of pyruvate kinase, and insulin expression was reduced in Pdx-1 ؉/؊ ͞Hnf-1␣ ؉/؊ mice. The islet cell architecture was also abnormal in Pdx-1 ؉/؊ ͞Hnf-3 ؉/؊ and Pdx-1 ؉/؊ ͞Hnf-1␣ ؉/؊ mice, with glucagon-expressing cells scattered throughout the islet, a defect that may be connected to decreased E-cadherin expression. Our data suggest that functional interactions between key islet regulatory factors play an important role in maintaining islet architecture and -cell function. These studies also established polygenic mouse models for investigating the mechanisms contributing to -cell dysfunction in diabetes.hepatocyte nuclear factors ͉ diabetes mellitus ͉ insulin secretion ͉ pancreatic islets G enetic and biochemical studies in humans and mice have identified many key transcription factors that are involved in controlling pancreas development and Ϫcell function in the adult islet (see ref. 1 for review). These factors have in common an islet-enriched expression pattern during pancreatic development, although each [e.g., hepatocyte nuclear factors (HNFs)] are also expressed in extrapancreatic tissues (e.g., intestine, liver, brain͞neurons, and kidney). Heterozygous mutations in a subset of these genes, including HNF-4␣, HNF-1␣, PDX-1, HNF-1, and BETA2, are specifically associated with a form of type 2 diabetes, termed maturity-onset diabetes of the young (MODY), that is characterized by an early disease onset (usually Ͻ25 years), autosomal dominant inheritance, and a primary defect in insulin secretion (see ref. 2 for review). There is increasing evidence that these and other islet-enriched transcription factors (e.g., Nkx2.2, Nkx6