Previous studies on the MHC class-specific differentiation of CD4(+)CD8(+) thymocytes into CD4(+) and CD8(+) T cells have focused on the role of coreceptor molecules. However, CD4 and CD8 T cells develop according to their MHC class specificities even in these mice lacking coreceptors. This study investigated the possibility that lineage is determined not only by coreceptors, but is also guided by the way how MHC molecules are presented. MHC class II molecules possess a highly conserved Cys in their transmembrane domain, which is palmitoylated and thereby associates with lipid rafts, whereas neither palmitoylation nor raft association was observed with MHC class I molecules. The generation of CD4 T cells was impaired and that of CD8 T cells was augmented when the rafts on the thymic epithelial cells were disrupted. This was due to the conversion of MHC class II-specific thymocytes from the CD4 lineage to CD8. The ability of I-A(d) molecule to associate with rafts was lost when its transmembrane Cys was replaced. The development of DO11.10 thymocytes recognizing this mutant I-A(dm) was converted from CD4 to CD8. These results suggest that the CD4 lineage commitment is directed by the raft-associated presentation of MHC class II molecules.