Significance Mucosa-associated invariant T (MAIT) cells are unconventional innate-like T cells recognizing microbial riboflavin metabolites presented by the monomorphic MR1 molecule. Here, we show that the CD8 ⁺ CD4 ⁻ and CD8 ⁻ CD4 ⁻ subpopulations of human MAIT cells represent transcriptionally and phenotypically discrete subsets with distinct functional profiles. Furthermore, T cell receptor repertoire analysis, as well as MAIT cell data based on human fetal tissues, umbilical cord blood, and culture systems indicate that the CD8 ⁻ CD4 ⁻ subset may derive from the main CD8 ⁺ CD4 ⁻ MAIT cell pool. Thus, MAIT cells, a major antimicrobial effector T cell population in humans, segregate into two functionally distinct but developmentally related subsets separated by the expression of CD8. This functional difference may have significant implications in infectious and inflammatory diseases.