CTLA-4-deficient mice develop a lethal autoimmune lymphoproliferative disorder that is strictly dependent on in vivo CD28 costimulation. Nevertheless, it is not known whether there is a specific site on the CD28 molecule that is required for induction of autoimmunity. Using CTLA-4-deficient mice expressing CD28 molecules with various point mutations in the CD28 cytosolic tail, the present study documents that in vivo costimulation for induction of autoimmune disease strictly requires an intact C-terminal proline motif that promotes lymphocyte-specific protein tyrosine kinase Lck binding to the CD28 cytosolic tail, because point mutations in C-terminal proline residues (Pro-187 and Pro-190) completely prevented disease induction. In contrast, in vivo costimulation for disease induction did not require either an intact YMNM motif or an intact N-terminal proline motif, which, respectively, promote phosphoinositide 3-kinase and IL2-inducible T cell kinase binding to the CD28 cytosolic tail. Thus, in vivo CD28 costimulation for induction of autoimmune disease is strictly and specifically dependent on an intact C-terminal proline motif that serves as a lymphocyte-specific protein tyrosine Lck kinase binding site in the CD28 cytosolic tail.