Inefficient thymic negative selection of self-specific T cells is associated with several autoimmune diseases, including type 1 diabetes. The factors that influence the efficacy of thymic negative selection, as well as the kinetics of thymic output of autoreactive T cells remain ill-defined. We investigated thymic production of β cell–specific T cells using a thymus-transplantation model. Thymi from different aged NOD mice, representing distinct stages of type 1 diabetes, were implanted into NOD. scid recipients, and the diabetogenicity of the resulting T cell pool was examined. Strikingly, the development of diabetes-inducing β cell–specific CD4+ and CD8+ T cells was regulated in an age-dependent manner. NOD. scid recipients of newborn NOD thymi developed diabetes. However, recipients of thymi from 7-and 10-d-old NOD donor mice remained diabetes-free and exhibited a progressive decline in islet infiltration and β cell–specific CD4+ and CD8+ T cells. A similar temporal decrease in autoimmune infiltration was detected in some, but not all, tissues of recipient mice implanted with thymi from NOD mice lacking expression of the autoimmune regulator transcription factor, which develop multiorgan T cell–mediated autoimmunity. In contrast, recipients of 10 d or older thymi lacked diabetogenic T cells but developed severe colitis marked by increased effector T cells reactive to intestinal microbiota. These results demonstrate that thymic development of autoreactive T cells is limited to a narrow time window and occurs in a reciprocal manner compared with colonic microbiota–responsive T cells in NOD mice.