Defects in pancreatic β-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) -1α, -3β, -4α, and Pdx-1 contribute in the complex transcriptional circuits within the pancreas that are involved in β-cell development and function. In mice, a heterozygous mutation in Pdx-1 alone, but not Hnf-1α^+/−, Hnf-3β^+/−, or Hnf-4α^+/−, causes impaired glucose-stimulated insulin secretion in mice. To investigate the possible functional relationships between these transcription factors on β-cell activity in vivo, we generated mice with the following combined heterozygous mutations: Pdx-1^+/−/Hnf-1α^+/−, Pdx-1^+/−/Hnf-3β^+/−, Pdx-1^+/−/Hnf-4α^+/−, Hnf-1α^+/−/Hnf-4α^+/−, and Hnf-3β^+/−/Hnf-4α^+/−. The greatest loss in function was in combined heterozygous null alleles of Pdx-1 and Hnf-1α (Pdx-1^+/−/Hnf-1α^+/−), or Pdx-1 and Hnf-3β (Pdx-1^+/−/Hnf-3β^+/−). 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 β-cell function in Pdx-1^+/−/Hnf-3β^+/− 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-1α^+/− mice. The islet cell architecture was also abnormal in Pdx-1^+/−/Hnf-3β^+/− and Pdx-1^+/−/Hnf-1α^+/− 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 β-cell function. These studies also established polygenic mouse models for investigating the mechanisms contributing to β-cell dysfunction in diabetes.