Abstract Mucosal-associated invariant T (MAIT) cells are a unique lineage of T cells that develop in the thymus. MAIT cells are positively selected by TCR recognition of microbial metabolite / MHC Class I-like molecule MR1 complexes presented on CD4/CD8 double-positive thymocytes. The developmental program of MAIT cells has been characterized as similar to that of invariant Natural Killer T (NKT) cells. Both MAIT and NKT cells acquire innate-like effector phenotypes during thymic development, and in each case differentiate into multiple subsets, with mature NKT cells categorized as either NKT1, NKT2 or NKT17, and mature MAIT cells as MAIT1 or MAIT17. We previously found distinct B7/CD28 and CD40L/CD40 co-stimulatory requirements for the development of thymic NKT subsets in BALB/c mice. In the absence of B7/CD28 co-stimulation, all NKT subsets were decreased, while only the NKT2 subset was specifically abrogated when lacking CD40L/CD40 co-stimulation. In the present study, we investigated the co-stimulatory requirements for MAIT cell development. Interestingly, MAIT17 cells were significantly increased when lacking either B7 or CD40 co-stimulation, while MAIT1 development was largely unaffected. These results revealed distinct co-stimulatory requirements for the thymic development of MAIT and NKT subsets. We are currently investigating the underlying mechanisms leading to the increase of MAIT17 cells in the absence of co-stimulatory signals.