Both positive and negative selection of immature T cells rely on engagement of their antigen-specific receptors (TCR) by peptide in association with proteins encoded in the major histocompatibility complex (MHC) protein. The decision made between these two outcomes seems to be determined by the number of TCR engaged by peptide-MHC complexes. It has been unclear how such a mechanism can be reconciled with evidence that positive and negative selection occur in different thymic compartments and are mediated by different antigen-presenting cells (APCs). In this study we demonstrate that the level of class I MHC protein is 10-fold higher on thymic dendritic cells, which mediate the negative selection of immature T cells, than on thymic epithelial cells, which mediate for positive selection. We also demonstrate that as little as a 3-fold increase in the level of a particular cognate peptide-MHC ligand is sufficient to result in negative rather than positive selection. The results suggest that quantitative differences in the level of expression of class I MHC proteins on thymic epithelial and dendritic cells contribute to the opposing roles these cells play in forming the repertoire of mature class I MHC restricted (CD8+) T cells.