ABCB1/MDR-1/P-glycoprotein (Pgp) is an ABC transporter responsible for cancer cell multi-drug resistance. It is expressed in cytotoxic T lymphocytes (CTL). Eliminating sensitive cancer cells during high-dose chemotherapy can also damage immune cells. Our study aimed to assess which maturing human CD8 + CTL memory subsets may be affected based on their Pgp protein expression. In an in vitro CTL differentiation model system, we tracked the maturation of naive, effector, and memory cells and the expression of Pgp. This system involves co-culturing blood lymphocytes with proliferation-inhibited JY antigen-presenting B-lymphoblastoid cells expressing HLA-I A2. These JY-primed maturing CTLs were TCR-activated using beads, and the effect of the maturation-modifying JAK1/2 inhibitor ruxolitinib was examined. Multidimensional analysis identified six major CTL subsets: naive, young memory (Tym), stem cell memory (Tscm), central memory (Tcm), effector memory (Tem), and effectors (Te). These subsets were further divided into thirteen specific subsets: TymCD127 + , TymCD127-, Tscm, TcmCD95 + , TcmCD73 +CD95 + , TcmCD95+CD127 + , TcmPD1 + , TemCD95 + , TemraCD127 + , TemraCD127-, TeCD95 + , and TeCD73 +CD95 + . Pgp expression was detectable in naïve cells and dynamically changed across the thirteen identified subsets. Increased Pgp was detected in young memory T cells and in Tscm, TcmCD95 + , and TcmPD1 + human CTL subsets. Unlike other transiently appearing memory cells, the number of cells in these core Pgp-expressing memory subsets stabilized by the end of the contraction phase. Ruxolitinib treatment downregulated effector T-cell polarization while upregulating small memory subsets expressing Pgp. In conclusion, activation increased Pgp expression, whereas ruxolitinib treatment preserved small early and late memory subset core that primarily expressed Pgp.