T cells play key roles in the immune system by detecting microbial molecules associated with infection. Mucosal-associated-invariant T (MAIT) cells represent between 5 and 50% of T cells in different human tissues and detect microbial vitamin-B2 (riboflavin) derivatives presented by the antigen-presenting molecule, MR1. They typically express an invariant TCR-a chain comprising TRAV1-2 joined to TRAJ33 genes, paired with a limited array of TRBV genes. We have identified three distinct developmental stages and transitional checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and 2 MAIT cells predominate in the thymus, while stage 3 cells progressively increase in percentage in cord blood, young, and adult peripheral blood. RNAseq analysis of separate developmental stages in mice and humans, has revealed marked transcriptional changes as MAIT cells mature which is allowing us to identify key factors that control each developmental checkpoint. We have also identified new populations of MR1-restricted T cells, some that utilize diverse TCR-ab gene usage and some that express gdTCRs, with distinct antigen specificity, including a population that binds derivatives of vitamin-B9 (folic acid) rather than vitamin-B2. Accordingly, our work demonstrates a high level of diversity in the MR1 restricted T cell repertoire, it maps their intrathymic developmental pathway, and identifies key checkpoints that control the generation of functional MAIT cells.