Cell culture methods that yield high numbers of functional antigen-specific T cells are crucial to translate adoptive T cell immunotherapy into clinics. In pre-clinical models, early differentiated memory T cell subsets showed superior engraftment, survival and function when compared to late differentiated T cell subsets. We aimed at the specific enrichment and preferential expansion of the distinctly early differentiated memory stem T cell subset (TSCM; CCR7+CD45RA+CD95+). In an explorative way, we established a Cytomegalovirus (CMV)-specific T cell culture system that investigates the influence of cytokine regimes, the addition of CD4+ T cells and the presence of regulatory T cells (TREG) on TSCM-derived T cell expansion. Here, we found that the expansion of antigen-specific TSCM-cells was diminished when bulk peripheral blood mononuclear cells were used as starting material. Specifically, we found that other memory T cell subsets inhibit the expansion of TSCM-cells. By the same token, we established that TSCM-cells expanded best when derived from pre-enriched CCR7+CD45RA+ T cells, cultured in the presence of irradiated bulk CD4+ T cells and distinct doses of IL-7 and IL-15. Surprisingly, depletion of TREG-cells significantly diminished TSCM-expansion irrespective of cytokines applied during culture. Further, post-stimulation CD45RO-selection owing to proliferation-induced CD45-isoform switch (CD45RA to CD45RO) significantly increased the purity and yield of TSCM-derived cultures. Our findings enable direct access to human antigen-specific TSCM-cells from peripheral blood and pave the way for rapid broad clinical application.