Summary Successful control of chronic virus and cancer critically depends on our improved understanding of the cellular and molecular mechanisms of T cell exhaustion. CD8 T cells responding to persistent antigens during chronic infection and cancer gradually differentiate into a dysfunctional state, commonly known as T cell exhaustion. Exhausted T cells acquire a unique and permanent epigenetic profile, which severely limits their functional reinvigoration. Recent studies from us and others reveal that the “exhausted” T cell pool consists of multiple phenotypically, functionally, and transcriptionally distinct subsets. Importantly, a TCF-1hi progenitor (TPRO) subset functions as a population of resource cells that continuously replenishes the pool of terminally exhausted T cells (TEXH) during chronic infection and cancer. Interestingly, we recently show that some TPRO cells can break away from the path toward exhaustion and differentiate into a CX3CR1+ effector subset (TEFF) with enhanced killing ability, which can be exploited as cellular therapies to control chronic viral infection and cancer in animal models. Furthermore, recent research demonstrates that the formation of these distinct CD8 T cell subsets is governed by underlying transcriptional programs. For example, TCF-1, T-bet and TOX act as subset-specific TFs to regulate the differentiation of TRPO, TEFF and TEXH, respectively. Perturbation of genetic pathways can be exploited to skew CD8 T cell differentiation trajectory toward effector subset and limit T cell exhaustion. Thus, the high-throughput discovery of novel transcription factors (TFs) that regulate the phenotypic heterogeneity of CD8 T cells and rapidly validate their biological function in the animal models is highly sought-after. Taking advantage of recently developed technologies in single cell sequencing and CRISPR/Cas9 gene editing, we propose to screen a set of novel TFs selected computationally based on their “regulon” activity in the respective subsets of CD8 T cells. This more targeted selection of candidate TFs combined with the high-throughput screen by perturb-seq will greatly expedite the discovery of novel transcriptional programs that regulate CD8 T cell differentiation and cell-fate choice. Furthermore, we will validate the function of the top-hits TFs identified by Perturb-seq via employing both loss-of-function and gain- of-function approaches to test the role of them in pathogen-specific CD8 T cells in vivo during chronic viral infection. The successful completion of this high-throughput, rapid and cost-effective CRISPR screen and functional validation of novel TFs in CD8 T cell differentiation will gain new knowledge in transcriptional regulation of T cell exhaustion. It will also provide mechanistic insights into therapeutic designs in treating cancer and infectious diseases.