We have developed a unique in vivo system to determine the relationship between endogenous altered peptide ligands and the development of major histocompatibility complex class II– restricted T cells. Our studies use the 3.L2 T cell receptor (TCR) transgenic mouse, in which T cells are specific for Hb(64–76)/I-E^k and positively selected on I-E^k plus self-peptides. To this endogenous peptide repertoire, we have individually added one of six well-characterized 3.L2 ligands. This transgenic approach expands rather than constrains the repertoire of self-peptides. We find that a broad range of ligands produce negative selection of thymocytes in vivo. When compared with the in vitro TCR–ligand binding kinetics, we find that these negatively selecting ligands all have a half-life of 2 s or greater. Additionally, one of two ligands examined with no detectable binding to the 3.L2 TCR and no activity on mature 3.L2 T cells (Q72) enhances the positive selection of transgenic thymocytes in vivo. Together, these data establish a kinetic threshold between negative and positive selection based on the longevity of TCR–ligand complexes.