Thymic positive selection is based on the interactions of T cell antigen receptors (TCRs) with self peptide–major histocompatibility complex (MHC) ligands, but the identity of selecting peptides for MHC class II–restricted TCRs and the functional consequences of this peptide specificity are not clear. Here we identify several endogenous self peptides that positively selected the MHC class II–restricted 5C.C7 TCR. The most potent of these also enhanced mature T cell activation, which supports the hypothesis that one function of positive selection is to produce T cells that can use particular self peptide–MHC complexes for activation and/or homeostasis. We also show that inhibiting the microRNA miR-181a resulted in maturation of T cells that overtly reacted toward these erstwhile positively selecting peptides. Therefore, miR-181a helps to guarantee the clonal deletion of particular moderate-affinity clones by modulating the TCR signaling threshold of thymocytes.