How lipidome changes support CD8+effector T (Teff) cell differentiation is not well understood. Here we show that, although naive T cells are rich in polyunsaturated phosphoinositides (PIPnwith 3–4 double bonds), Teffcells have unique PIPnmarked by saturated fatty acyl chains (0–2 double bonds). PIPnare precursors for second messengers. Polyunsaturated phosphatidylinositol bisphosphate (PIP2) exclusively supported signaling immediately upon T cell antigen receptor activation. In late Teffcells, activity of phospholipase C-γ1, the enzyme that cleaves PIP2into downstream mediators, waned, and saturated PIPnbecame essential for sustained signaling. Saturated PIP was more rapidly converted to PIP2with subsequent recruitment of phospholipase C-γ1, and loss of saturated PIPnimpaired Teffcell fitness and function, even in cells with abundant polyunsaturated PIPn. Glucose was the substrate for de novo PIPnsynthesis, and was rapidly utilized for saturated PIP2generation. Thus, separate PIPnpools with distinct acyl chain compositions and metabolic dependencies drive important signaling events to initiate and then sustain effector function during CD8+ T cell differentiation.