The development of αβ T-cells is a step-wise process guided by unique stromal microenvironments within the thymus, which results in the formation of T-cells with a highly diverse repertoire of T-cell receptors (TCRs). In the latter stages of development, T-cell tolerance is established through the selective deletion of cells expressing auto-reactive TCRs, in addition to the generation of immunosuppressive regulatory T-cells (T-Reg). Central tolerance induction is mediated by interaction with functionality heterogeneous medullary thymic epithelial cells (mTEC). The aim of this study was to search for a novel mTEC-expressed functional molecules involved in medullary homeostasis and central tolerance. Here we identify two novel mTEC populations; cells expressing osteoprotegerin (OPG), a negative regulator of Rank-mediated mTEC maturation, and inducible nitric oxide synthase (iNOS), an immune-active enzyme catalysing the production of nitric oxide. Importantly, we found OPG to restrict the size of the mTEC compartment, which in contrast to previous findings had no impact on T-Reg production, and instead limited the influx of peripheral lymphocytes to the thymus. In contrast, iNOS appears to play only a minor role in the maturation of single positive thymocytes in the medulla. These findings highlight the importance of functionally distinct mTEC compartments in maintaining medullary homeostasis.