Memory stem T (TSCM) cells, a new subset of memory T cells with self-renewal and multipotent capacities, are considered as a promising candidates for adoptive cellular therapy. However, the low proportion of human TSCM cells in total CD8+ T cells limits their utility. Here, we aimed to induce human CD8+ TSCM cells by stimulating naive precursors with interleukin-21 (IL-21). We found that IL-21 promoted the generation of TSCM cells, described as CD45RA+CD45RO−CD62L+CCR7+CD122+CD95+ cells, with a higher efficiency than that observed with other common γ-chain cytokines. Upon adoptive transfer into an A375 melanoma mouse model, these lymphocytes mediated much stronger antitumor responses. Further mechanistic analysis revealed that IL-21 activated the Janus kinase signal transducer and activator of transcription 3 pathway by upregulating signal transducer and activator of transcription 3 phosphorylation and consequently promoting the expression of T-bet and suppressor of cytokine signaling 1, but decreasing the expression of eomesodermin and GATA binding protein 3. Our findings provide novel insights into the generation of human CD8+ TSCM cells and reveal a novel potential clinical application of IL-21.