Mucosal-associated-invariant T (MAIT) cells detect microbial vitamin-B2 derivatives presented by the antigen-presenting molecule, MR1, and represent the largest population of abT cells with a single specificity within the human body. Yet, MAIT cell numbers vary widely between individuals, thus it is important we understand the factors that regulate their development and function. Here we map the developmental pathway by identifying new populations of thymic MAIT cells in humans and mice, and the multiple checkpoints that control the generation of functional MAIT cells^#. Transition through each checkpoint is regulated by MR1 and the final checkpoint that generates mature functional MAIT cells is controlled by multiple factors, including the transcription factor PLZF and microbial colonisation. MAIT cell maturation can also occur after thymic emigration of immature MAIT cells. We proceed to examine these MAIT cell maturation subsets from mice and humans, by RNA-sequencing in combination with flow cytometry and real time PCR to decipher the factors that are critical for this progression. In addition to validating the known cell surface profile, we also demonstrate the regulation of MAIT cells by key genetic factors involved in modulating T cell development. Notably, there is sharp downregulation of LEF1 and SATB1; and upregulation Id2, IL23R, and ICOS; between stage 1 and 3 of MAIT cell maturation. We go on to demonstrate that several of these factors play a significant role in MAIT cell maturation, and provide detailed transcriptome analysis to the mechanisms in which MAIT cells develop. Accordingly, our study maps the developmental pathway and checkpoint candidates that control the generation and variations in the numbers of functional MAIT cells.