Va24-invariant NKT cells (NKTs) effectively localize to tumors and have natural antitumor properties that make them highly attractive as a carrier of chimeric antigen receptors (CARs) for redirected cancer immunotherapy. However, the mechanisms responsible for NKT-cell numeric expansion and in vivo persistence are unknown, impeding the clinical application of NKTs or CAR-NKTs. We show that antigen-induced in vitro expansion of primary NKTs is associated with the accumulation of a CD62L+ subset, while CD62L- cells undergo early exhaustion. Only CD62L+ NKTs survived and proliferated in response to secondary stimulation. After transfer to NSG mice, CD62L+ NKTs persisted 5 times longer than CD62L- NKTs and when transduced with a CD19-specific CAR had higher therapeutic efficacy in a Raji lymphoma model. Proliferating CD62L+ cells downregulated or maintained CD62L expression when they were activated via TCR alone or in combinations with co-stimulatory receptors, respectively. We then tested 161 clones of K562 cells genetically modified to express CD1d and co-stimulatory ligands. We found that the B-8-2 clone (HLA^nullCD1d^medCD86^high4-1BBL^medOX40L^high) induced the highest rate of NKT-cell expansion with the preservation of CD62L expression. Compared with CAR-NKTs expanded with autologous PBMC, B-8-2-expanded cells exhibited prolonged in vivo persistence and superior therapeutic activity in a Raji lymphoma model. Therefore, we have identified CD62L as a marker of a functionally distinct NKT-cell subset endowed with high proliferative potential and have developed artificial antigen-presenting cells for the generation of CD62L-enriched NKTs for effective cancer immunotherapy.