Vα24-invariant Natural Killer T cells (NKTs) have potent antitumor properties and are being developed for cellular immunotherapy of cancer. Such therapy requires extensive ex vivo expansion of primary NKTs while preserving their longevity and function. In a recent report from our group, CD62L⁺ subset of NKTs has been shown to have longer persistence in vivo and stronger antitumor activity than CD62L^- counterpart. However, the requirements for the preservation of CD62L⁺ NKTs during ex vivo expansion remain largely unknown. Comparative gene expression analysis of CD62L⁺ and CD62L^- NKT subsets revealed a significantly higher expression of IL-21R in the former, which was confirmed at the protein level by flow cytometry. Hence, we hypothesized that IL-21 preferentially supports CD62L⁺ NKTs. To test this hypothesis, we expanded primary human peripheral blood NKTs using in vitro stimulation with their cognate antigen, α-galactosylceramide. The culture was supplemented with IL-2, IL-21, or both cytokines. We found that in contrast to IL-2, IL-21 alone failed to support NKT-cell expansion. However, a combined treatment with IL-2 and IL-21 produced more NKTs compared with IL-2 alone. Moreover, the former condition significantly increased frequency of CD62L⁺ NKTs that was associated with the selective downregulation of a pro-apoptotic gene BCL2L11 in the CD62L⁺ NKT-cell subset. We also found that IL-2/IL-21-expanded NKTs were more cytotoxic against lymphoma cells that correlated with enhanced expression of granzyme B. Following adoptive transfer to NSG mice, IL-2/IL-21-expanded NKTs persisted significantly longer and had higher therapeutic efficacy in a lymphoma model compared with IL-2-expanded NKTs. Our results instruct inclusion of IL-21 in the NKT-cell expansion protocols for cancer immunotherapy applications.