Combining CD5-null, MHC-deficient and lineage-specific reporter animals, we have investigated the influence of CD5 on positive selection and the choice of CD4- versus CD8-lineage commitment on broad populations of thymocytes. CD5 has no obvious quantitative effect in wild-type mice. In mice lacking MHC class II molecules, however, increased numbers of transitional, class I-selected CD4+ CD8(int) CD3(hi) cells were positively selected in the absence of CD5. Importantly, they were committed to the CD4 lineage. Our results indicate that CD5 negatively regulates the differentiation of CD4-committed cells in suboptimal conditions, thus perhaps serving to tighten the correlation between restriction of the TCR and lineage choice.