Natural thymic T regulatory (tTreg) cells maintain tolerance to self-antigen. These cells are generated in the thymus, but how this generation occurs is still controversial. Furthermore, the contribution of thymus epithelial cells to this process is still unclear, especially in humans. Using an exceptional panel of human thymic samples, we demonstrated that medullary thymus epithelial cells (mTECs) promote the generation of tTreg cells and favor their function. These effects were mediated through soluble factors and were mTEC specific since other cell types had no such effect. By evaluating the effects of mTECs on the absolute number of Treg cells and their state of proliferation or cell death, we conclude that mTECs promote the proliferation of newly generated CD25+ cells from CD4+CD25- cells and protect Treg cells from cell death. This observation implicates Bcl-2 and mitochondrial membrane potential changes, indicating that the intrinsic cell death pathway is involved in Treg protection by mTECs. Interestingly, when the mTECs were cultured directly with purified Treg cells, they were able to promote their phenotype but not their expansion, suggesting that CD4+CD25- cells have a role in the expansion process. To explore the mechanisms involved, several neutralizing antibodies were tested. The effects of mTECs on Treg cells were essentially due to interleukin (IL)-2 overproduction by thymus CD4+ T cells. We then searched for a soluble factor produced by mTECs able to increase IL-2 production by CD4+ cells and could identify the inducible T-cell costimulator ligand (ICOSL). Our data strongly suggest a « ménage à trois »: mTEC cells (via ICOSL) induce overproduction of IL-2 by CD25- T cells leading to the expansion of tTreg cells. Altogether, these results demonstrate for the first time a role of mTECs in promoting Treg cell expansion in the human thymus and implicate IL-2 and ICOSL in this process.