Adoptive T-cell immunotherapy provides a promising approach to cancer therapy. The persistence and resistance to exhaustion of transferred T cells are critical for improvement in patient outcomes. Stem cell memory T (TSCM) cells have been proposed as a new class of memory T cells which have longevity and proliferative potential. It has been shown that mouse and human CD8+ TSCM cells can be generated in vitro from naïve CD8+ T cells by the Wnt signaling, however, the methods for inducing TSCM cells from activated or memory T cells remain to be established. Here, we established a new strategy of generating TSCM-like cells in vitro (designated as “iTSCM” cells) from activated CD4+ and CD8+ T cells in mice and humans by coculturing with stromal cells expressing a Notch ligand. These iTSCM cells lost PD-1 and CTLA-4 expression, were resistant to cell cycle arrest and apoptosis, and produced a large number of effector cells after restimulation, therefore exhibiting a strong antitumor activity. The present study illustrates a novel approach of generating a large number of antigen-specific effector cells for T cell-based adoptive immunotherapy.