Abstract Innate lymphoid cells (ILCs) play diverse roles in shaping innate and adaptive immunity. These cells exist as heterogeneous populations and their identities are influenced by their tissue environments. Likewise, the ontogeny of ILCs is also complex. Although ILCs are thought to arise from bone marrow progenitors or tissue resident progenitors distributed during embryogenesis, work from our laboratory strongly suggest that ILCs at least ILC2s can also be generated in the thymus, not only from multipotent progenitors but also from committed T cell precursors. Our recent data using single cell RNA sequencing suggest that a substantial fraction of the ILC-enriched population in the blood of wild type mice (WT) come from the thymus, and their equivalents can also be found in peripheral tissues. We thus hypothesize that the thymus exports a substantial amount of ILC precursors to the circulation, which may replenish ILC2s and/or other ILCs in peripheral tissues in steady-state or upon immune challenges and the thymus-derived ILCs may have distinct functions. In this renewal proposal, we intend to follow up on these new exciting findings and determine the function of these thymus-derived ILC precursors and their biological significant. We will further characterize thymus-derived ILC-precursors in the lung and small intestine and determine their frequencies during mouse development. We will also identify thymus-derived ILC precursors in human blood, thus gaining appreciation of the clinical relevance of these cells. We will then focus on learning the function of these thymus-derived ILC precursors using in vitro and in vivo approaches, as well as adoptive transfer of purified ILC precursors into Rag2-/-Il2g-/- mice which are devoid of ILCs. The behaviors of these cells in type 2 immune reactions will be monitored. Finally, we will assess the cell-intrinsic functional differences among WT ILC2s generated from bone marrow common lymphoid progenitors (CLP) and thymic DN1 and DN3 T cell precursors. Taken together, studies outlined in this proposal will further our understanding of thymus-derived ILCs, which will help establish a new paradigm regarding to the production and maintenance of ILC pools. Because the presence of an active thymus represents one of the major differences between children and adults, knowledge about thymus-derived ILCs may shed light on their different immune responses such as those during Covid-19 infection.