Major histocompatibility complex-related protein 1 (MR1) is a highly conserved non-polymorphic antigen presenting protein that is well known for the presentation of microbially-derived riboflavin metabolites to mucosal-associated invariant T (MAIT) cells. Once thought to be limited to this one modality, recent work has shown that MR1 and the T cells that recognize it are much more diverse than previously appreciated. Most notably, recent reports of MR1-restricted T cell (MR1T) clones that have shown MR1-dependent cytotoxic activity against cancerous tissues has opened the door for the study of potential endogenous ligands for MR1. The work described in this dissertation provides context for these recent developments, positing that the newer “atypical” members of the MR1T family may be more unified in their recognition of MR1 both in homeostasis and in the context of cancer. My characterization of a new subset of endogenously derived MR1 ligands, endogenous pterin (EP) molecules, answers the outstanding questions around the ability of MR1 to present “self” metabolites. Further investigation of these molecules reveals that they can activate cancer-associated MR1Ts, but beyond this cancer paradigm, I also show that these same ligands can activate 6-FP reactive and “atypical” non-cancer-associated MR1Ts. This implies that these unmodified endogenous metabolites may play a much broader role in MR1T biology, perhaps explaining the selection of these cells in the thymus or their maintenance in the periphery. Finally, I show how previously discovered microbial ligands for MR1 can be used to define new populations of MR1T cells, introducing the first instance of antigen specific recognition of MR1 by a γδ T cell. Altogether, this work emphasizes the functional versatility of MR1, and through the discovery of new ligand classes, and the reassessment of others, it provides answers for some of the major outstanding questions surrounding the biology of MR1T cells.