Natural CD4 + CD25 + regulatory T (CD4 + CD25 + T reg) cells play a key role in the immunoregulation of autoimmunity. However, little is known about the interactions between CD4 + CD25 + T reg cells and autoreactive T cells. This is due, in part, to the difficulty of using cell surface markers to identify CD4 + CD25 + T reg cells accurately. Using a novel real-time PCR assay, mRNA copy number of FoxP3, TGFβ1, and interleukin (IL)-10 was measured in single cells to characterize and quantify CD4 + CD25 + T reg cells in the nonobese diabetic (NOD) mouse, a murine model for type 1 diabetes (T1D). The suppressor function of CD4 + CD25 + CD62L hi T cells, mediated by TGFβ, declined in an age-dependent manner. This loss of function coincided with a temporal decrease in the percentage of FoxP3 and TGFβ1 coexpressing T cells within pancreatic lymph node and islet infiltrating CD4 + CD25 + CD62L hi T cells, and was detected in female NOD mice but not in NOD male mice, or NOR or C57BL/6 female mice. These results demonstrate that the majority of FoxP3-positive CD4 + CD25 + T reg cells in NOD mice express TGFβ1 but not IL-10, and that a defect in the maintenance and/or expansion of this pool of immunoregulatory effectors is associated with the progression of T1D.