The study investigated the interaction between ruxolitinib (RUX), an FDA-approved JAK1/2 inhibitor, and P-glycoprotein (Pgp/ABCB1/MDR1), an ATP-binding cassette transporter involved in drug resistance and immune regulation. Pgp exports xenobiotics and is expressed in immune cells, including T lymphocytes. While high Pgp expression is linked to poor responses in autoimmune diseases, it enhances the resilience of memory T cells, supporting immune recovery after chemotherapy. To evaluate this interaction, human primary T cells and NIH-3T3 MDR1 cells were used. RUX inhibited Pgp activity in a dose-dependent manner (at non-therapeutic levels), downregulated PD-1 and Pgp in activated T cells, activated basal Pgp ATPase activity, and interfered with verapamil-induced activation, suggesting functional modulation. Molecular analyses revealed that ABCB1 mRNA was high in unprimed T cells but declined with activation. RUX treatment upregulated ABCB1 in activated T cells, indicating its role as a transcriptional regulator. These findings may improve chemotherapy outcomes and aid in GVHD management. The effect of RUX on T-cell maturation showed a shift toward naive and central memory (Tcm) CD8+ T cells, with reduced effector and effector memory (Tem) subsets. This reprogramming may enhance therapeutic efficacy in myelofibrosis and immune disorders. Using an in vitro model, T cells were primed with JY antigen-presenting cells, and maturation was tracked over a month. RUX exposure promoted Pgp+ long-lived memory T cell maintenance and delayed effector differentiation. It also expanded CD127⁺ memory T cells, supporting long-term immune memory. In conclusion, this study demonstrates that RUX modulates Pgp function and T cell differentiation, with implications for autoimmune disease, transplantation, and cancer therapy. Targeting Pgp in memory T cells could improve the effectiveness of immunotherapies by enhancing memory cell survival and immune resilience.