Regulatory T cells (Tregs) are part of the suppressive arm of the immune system, playing an important role in maintaining immune homeostasis and preventing excessive inflammatory responses. As autoimmune diseases become increasingly prevalent, it is important to clarify the role of Tregs in etiology and their potential for use in cell therapy. Characterization of Tregs has revealed specialized subsets that have unique functions in controlling tissue-specific inflammation. Of particular interest is the distinction between Tregs that differentiate within the thymus (tTregs) versus the periphery (pTregs). While tTregs recognize self-antigen, pTregs are generated to induce tolerance to non-self-antigens such as those from commensal microbiota and self-antigens not presented in the thymus. pTregs could play a unique non-redundant role in type 1 diabetes (T1D), a chronic autoimmune disease, based on recent work showing connections between microbiota and T1D incidence as well as the generation of neoantigens within the pancreas. Therefore, we created a new model to determine the importance of pTregs in T1D by deleting a key genetic enhancer of Foxp3 for pTreg generation (conserved noncoding sequence CNS1) within the autoimmune prone non-obese diabetic (NOD) mouse. CNS1 deletion does not alter Treg frequencies or characteristics in young female NOD mice in most organs except the large intestine lamina propria, where increased Helios expression and reduced RORt expression suggest a shift towards tTregs. Despite increased insulitis, these mice do not exhibit increased T1D incidence. Surprisingly, we also observed that Tregs in the islet do not recognize peripherally-derived neoantigens made from insulin fusion products, and the reactivity of Tregs and Tconvs is not changed in prediabetic NOD CNS1-/- females. Lastly, preliminary data suggest NOD CNS1-/- females do not show defects in maternal-fetal tolerance but could exhibit significantly reduced Treg frequencies in the spleen and increased frequencies in the pancreatic and mesenteric lymph nodes. We concluded that CNS1-dependent pTregs do not play a significant role in the NOD mouse model, with tTregs being the key regulators of T1D. Additionally, Tregs within pancreatic islets largely recognize thymically-presented antigens, and pTregs are not frequently generated against pancreatic neoantigens.