T cells reactive to β cell Ags are critical players in the development of autoimmune type 1 diabetes. Using a panel of diabetogenic CD4 T cell clones derived from the NOD mouse, we recently identified the β cell secretory granule protein, chromogranin A (ChgA), as a new autoantigen in type 1 diabetes. CD4 T cells reactive to ChgA are pathogenic and rapidly transfer diabetes into young NOD recipients. We report in this article that NOD. ChgA−/− mice do not develop diabetes and show little evidence of autoimmunity in the pancreatic islets. Using tetramer analysis, we demonstrate that ChgA-reactive T cells are present in these mice but remain naive. In contrast, in NOD. ChgA+/+ mice, a majority of the ChgA-reactive T cells are Ag experienced. Our results suggest that the presence of ChgA and subsequent activation of ChgA-reactive T cells are essential for the initiation and development of autoimmune diabetes in NOD mice.