T cell activation, proliferation, and differentiation involve metabolic reprogramming resulting from the interplay of genes, proteins, and metabolites. Here, we aim to understand the metabolic pathways involved in the activation and functional differentiation of human CD4+ T cell subsets (T helper [Th]1, Th2, Th17, and induced regulatory T [iTreg] cells). Here, we combine genome-scale metabolic modeling, gene expression data, and targeted and non-targeted lipidomics experiments, together with in vitro gene knockdown experiments, and show that human CD4+ T cells undergo specific metabolic changes during activation and functional differentiation. In addition, we confirm the importance of ceramide and glycosphingolipid biosynthesis pathways in Th17 differentiation and effector functions. Through in vitro gene knockdown experiments, we substantiate the requirement of serine palmitoyltransferase (SPT), a de novo sphingolipid pathway in the expression of proinflammatory cytokines (interleukin [IL]-17A and IL17F) by Th17 cells. Our findings provide a comprehensive resource for selective manipulation of CD4+ T cells under disease conditions characterized by an imbalance of Th17/natural Treg (nTreg) cells.