Type I diabetes (T1D) is an autoimmune disease characterized by the destruction of pancreatic islet cells primarily by CD4+ and CD8+ T cells that escape mechanisms of self-tolerance. During T cell development in the thymus, negative selection eliminates autoreactive cells from the repertoire. This process requires the transcription factor, autoimmune regulator (AIRE), which promotes the expression of peripheral self-antigens within the thymus. In humans with T1D and in the nonobese diabetic (NOD) mouse model of the disease, insulin is a major autoantigen detected by pathogenic T cells, and Aire controls insulin expression in the thymus. Modeling studies suggest insulin can be presented to T cells in complex with the IAg7 major histocomaptibility complex (MHC) in three possible binding registers. Previous data suggests that many T cell clones isolated from the pancreas, unexpectedly, recognize the most energetically unfavorable binding confirmation, register 3. In agreement, it has been proposed that insulin peptide may be loaded directly into IAg7 on the surface of APCs in the islet, bypassing traditional antigen presentation pathways that would prohibit the display of an unfavorable binding register. We propose that most register 1 and 2-specific T cells are deleted in the thymus via expression of insulin by Aire, whereas register 3-reactive T cells escape negative is not efficiently presented in the islet. selection. To test this, we generated a TCR-transgenic mouse from a T cell hybridoma, 4F7, that recognizes insulin. Interestingly, this T cell clone is weakly diabetogenic but appears to induce severe insulitis. Furthermore, analysis of 4F7+ T cell development in the absence of Aire and insulin suggests that Aire plays a role in the negative selection Insulin-specific T cells but in subtle ways that were not previously appreciated. Taken together these studies help shed further light on mechanisms that keep insulin reactive T cells in check.