T cells play key roles in the immune system by detecting microbial peptides associated with infection. However, one major T cell population, mucosal-associated invariant T (MAIT) cells, are activated by microbial vitamin B2 (riboflavin) derivatives presented by the major histocompatibility complex (MHC) class I related protein, MR1. In humans, MAIT cells represent between 5 and 50% of T cells in different human tissues. The numbers of MAIT cells can vary widely between individuals and the factors that govern the development of these cells remain elusive. Using MR1 tetramers to detect MAIT cells in mice and humans, we have now identified thymic precursors to the MAIT cell lineage and delineated three distinct stages in MAIT cell development. In mice, the least mature, stage 1 (CD24⁺CD44^-) cells progress to an intermediate stage 2 (CD24^-CD44^-) before maturing into stage 3 (CD24^-CD44⁺) MAIT cells. Progression through each of these checkpoints is MR1 dependent, while the final maturation checkpoint (from stage 2 to 3) that gives rise to functional MAIT cells requires the transcription factor, promyelocytic leukemia zinc finger (PLZF). In humans, stage 1 cells (CD161^-CD27^-) and stage 2 cells (CD161^- CD27⁺) predominate in the thymus, while stage 3 cells (CD161⁺) progressively increase in percentage in umbilical cord blood, young peripheral blood and adult peripheral blood. Thus, MAIT cell maturation can also occur after thymic emigration of immature MAIT cells in humans and akin to MAIT cell development in mice, human MAIT cells acquire PLZF as they advance from stage 1 to 3. Accordingly, our study maps the intrathymic developmental pathway and identifies key checkpoints that control the maturation of MAIT cells in mice and humans, which will underpin investigation of the factors that regulate numbers of these cells.