Clonal deletion of autoreactive thymocytes is important for self-tolerance, but the intrathymic signals that induce clonal deletion have not been clearly identified. We now report that clonal deletion during negative selection required CD28-mediated costimulation of autoreactive thymocytes at the CD4+CD8lo intermediate stage of differentiation. Autoreactive thymocytes were prevented from undergoing clonal deletion by either a lack of CD28 costimulation or transgenic overexpression of the antiapoptotic factors Bcl-2 or Mcl-1, with surviving thymocytes differentiating into anergic CD4?CD8? double-negative thymocytes positive for the T cell antigen receptor ?? subtype (TCR??) that 'preferentially' migrated to the intestine, where they re-expressed CD8? and were sequestered as CD8??+ intraepithelial lymphocytes (IELs). Our study identifies costimulation by CD28 as the intrathymic signal required for clonal deletion and identifies CD8??+ IELs as the developmental fate of autoreactive thymocytes that survive negative selection. [ABSTRACT FROM AUTHOR]