Program Summary/Abstract Immunotherapies including adoptive cell therapy (ACT) and immune checkpoint blockade (ICB) represent powerful approaches in combating cancer. CD8+ cytotoxic T lymphocytes (CTLs) are a cornerstone of cancer immunity and exhibit inherent heterogeneity, with precursor exhausted T (Tpex) but not exhausted T (Tex) cells responding to immunotherapies. However, poor functional and proliferative capacity of CTLs in the tumor microenvironment limit immunotherapeutic efficacy. Further, whether and how Tex cells, which retain limited cytolytic activity against tumors, can be reprogrammed to reinvigorate anti-tumor immunity remains largely unknown. Thus, there is an urgent need to systemically interrogate the regulatory circuitry underlying CTL differentiation to unleash the full immunotherapeutic potentials. Through single-cell CRISPR (scCRISPR) screens of CD8+ T cells in vivo, we discovered two transcriptional axes, Rbpj–Irf1 and Ets1–Batf, that impinge upon CTL differentiation states and metabolic activities. Targeting Rbpj inhibits transitory Tex (Tex1) to terminal Tex (Tex2) cell differentiation, while targeting Ets1 promotes Tpex to transitory Tex (Tex1) cell differentiation. Accordingly, targeting Rbpj or Ets1 enriches Tex1 cells, associated with enhanced metabolic activities, and shows strong therapeutic efficacy in multiple murine tumor models. Low RBPJ or ETS1 expression strongly correlates with the immunotherapeutic response in human patients with cancer, further supporting the therapeutic relevance. Our central hypothesis is that CTL differentiation is marked by transcriptional and metabolic heterogeneity, the targeting of which enriches Tex1 cells and promotes antitumor immunity. Our model represents a major advance towards understanding fundamental mechanisms of T cell fate and immunometabolism and points to new avenues to improve efficacy of cancer immunotherapies. Aim 1. Target mediators of Tex1 to Tex2 terminal differentiation to reinvigorate exhausted T cells. Aim 2. Establish mechanisms that control T cell metabolism and Tex1 cell generation. Aim 3. Identify combinatorial factors and metabolic dependence for CTL differentiation. We predict our studies will establish new paradigms in T cell biology and immuno-oncology, with the discovery of novel checkpoints and actionable targets to reprogram CTL cell fate by enriching the Tex1 state and rewiring cellular metabolism, thereby opening new avenues to improve cancer therapies.