Early T cell development requires activation of Notch receptors to regulate two distinct processes: progenitor self-renewal and cell lineage decisions. By modulating Notch activation during late stages of T cell development, we have uncovered novel and unexpected roles for Notch signaling in thymic differentiation of multiple innate/unconventional T cells. In contrast to conventional T cells, whose effector functions are programmed after thymic egress, innate T cell lineages develop with pre-programmed effector functions, such as cytokine production or cytoxicity, in the thymus. In this talk, I will illustrate how we have used advanced and highly dimensional single cell technologies, such as CyTOF and “multi-omics” immune profiling to better understand Notch-regulated innate T cell differentiation in the thymus.