The primary goal of this project is to map distinct hematopoietic progenitors during embryonic development that give rise to specific subsets of T cells and to understand how these progenitors influence T cell function and lineage diversity. This research specifically aims to explore how developmental origin shapes the phenotypic and functional characteristics of CD8+ T cells. The project has two main objectives: First, to create a high-resolution map of T cell lineages derived from different embryonic progenitors by isolating and culturing cells from various developmental stages (E9.5, E12.5, E15.5, and adult bone marrow) using the artificial thymic organoid (ATO) system. Advanced flow cytometry and single-cell sequencing will be used to characterize and map the resulting CD8+ T cell subsets, our labs focus. Second, the functional potential of CD8+ T cells from these progenitors will be assessed by evaluating their responses to TCR-dependent and TCR-independent signals through both in vitro stimulation and in vivo models. For the in vivo studies, progenitors will be transplanted into sub-lethally irradiated neonatal mice to study T cell development in a physiological setting. These mice will then be infected, and CD8+ T cell responses will be monitored to examine how hematopoietic origin influences T cell fate during infection. This research plan will be carried out during the 1-year funding period, providing revolutionary insights into the developmental origins of T cell diversity and their functional roles in immunity.