One of the central questions in immunology is how adaptive immunity generates its diversity while maintaining tolerance. The key mediators of tolerance are T cells, which develop in the thymus, where thymocytes rearrange their T cell receptor genes and undergo positive and negative selection. This ensures their ability to recognise antigen in the context of MHC, whilst avoiding self-reactivity. We are still far from fully understanding the steps in these processes at the molecular level, especially in humans: What are the developmental trajectories of different T cell subtypes, and how do they relate to their journey through the organ during maturation? What is the functional role played by the macro- and micro-scale environments in regulating this? These questions have gained in importance since several T cell types are now therapeutics in cancer and transplantation, raising the question of how to engineer specific T cell subsets in vitro. In AIM 1, we propose to generate an organ-scale 3D thymic cell atlas at full genomic breadth through genomics and imaging technologies. By combining multi-modal single cell genomics with multi-scale spatial genomics and imaging technologies, we will generate a rich data set for deep and comprehensive reconstruction of tissue architectures in a thymic lobe. In AIM 2, we will carry out computational data integration with new methods for 3D multi-modal atlas assembly to predict lymphocyte developmental mechanisms at micro and macro scales. In AIM 3, we will use an artificial thymic organoid system to simultaneously validate our findings and enhance T cell engineering approaches. This powerful integrated approach combines genomics, imaging and tissue engineering together with computational analyses to dissect design principles of the thymus, a central organ of the immune system. This knowledge will guide the development of engineered T cells as research reagents, and ultimately as therapeutics.