Defects in regulatory T cell (Treg) development and/or function are commonly believed to contribute to the loss of immune cell tolerance to islet antigens leading to type 1 diabetes (T1D). Conflicting data exists as to whether Treg numbers and function are reduced in individuals with T1D. In this study, we examine in detail the defects in Treg development and function in T1D prone NOD mice compared with various T1D protected strains including B6 and NOD congenic mice carrying protective alleles at Idd3 (Il2) or Idd5 (Ctla4, Scl11a1 and Acadl). As the Idd3 and Idd5 protective alleles interact to protect from diabetes, we tested whether these two regions synergistically improve Treg development and function. When compared with B6, NOD mice were found to have significantly reduced frequencies of Tregs in the lymph nodes and Peyer’s patches but not the spleen. Tregs in both strains expanded and persisted for an equivalent time period following IL-2/anti-IL-2 complex therapy. NOD mice had reduced Treg numbers in all peripheral lymphoid organs assessed compared with Idd3/5 mice. Both the Idd3 and Idd5 regions contributed to restoring Treg numbers. Thymic output of Tregs was equivalent between the strains suggesting that peripheral homeostasis was impacted. The proportion of Helios negative Tregs (marking peripherally induced Tregs) was not altered. Interestingly, Idd3 and Idd5 interacted to increase the expression of CD25 on Tregs. These findings demonstrate that genes associated with T1D interact resulting in significant defects in the Treg compartment. These defects can potentially be overcome with IL-2 therapy.