During their final maturation in the medulla, semimature single-positive (SP) thymocytes downregulate activation markers and subsequently exit into the periphery. Although semimature CD4(+) SP cells are sensitive to negative selection, the timing of when negative selection occurs in the CD8 lineage remains elusive. We show that the abundance of terminally matured CD8(+) SP cells in adult thymus is modulated by the genetic background. Moreover, in BALB/c mice, the frequency of terminally matured CD8(+) SP cells, but not that of CD4(+) SP cells present in thymus, varies depending on age. In mice lacking expression of the adhesion receptor CD155, a selective deficiency of mature CD8(+) SP thymocytes was observed, emerging first in adolescent animals at the age when these cells start to accumulate in wild-type thymus. Evidence is provided that the mature cells emigrate prematurely when CD155 is absent, cutting short their retention time in the medulla. Moreover, in nonmanipulated wild-type mice, semimature CD8(+) SP thymocytes are subjected to negative selection, as reflected by the diverging TCR repertoires present on semimature and mature CD8(+) T cells. In CD155-deficient animals, a shift was found in the TCR repertoire displayed by the pool of CD8(+) SP cells, demonstrating that CD155 is involved in negative selection.