In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8 T cell fate decisions.

How early events in effector T cell (T) subsets tune memory T cell (T) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of T and T cells using in vivo pooled CRISPR screening, focusing on negative regulators of T responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of T differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among T cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent T proliferation and T development. Our study establishes that nutrient uptake and signaling are key determinants of T cell fate and shape the quantity and quality of T responses.