Murine models showed that CD8⁺CD44^hi memory T (T_M) cells could eradicate malignant cells without inducing graft-versus-host disease (GVHD). We evaluated the feasibility and safety of infusing freshly isolated and purified donor-derived phenotypic CD8⁺ T_M cells into adults with disease relapse after allogeneic hematopoietic cell transplantation (HCT). Phenotypic CD8 T_M cells were isolated after unmobilized donor apheresis using a tandem immunomagnetic selection strategy of CD45RA depletion followed by CD8⁺ enrichment. Fifteen patients received CD8⁺ T_M cells at escalating doses (1 × 10⁶, 5 × 10⁶, or 10 × 10⁶ cells per kg). Thirteen received cytoreduction before CD8⁺ T_M cell infusion, and 9 had active disease at the time of infusion. Mean yield and purity of the CD8⁺ T_M infusion were 38.1% and 92.8%, respectively; >90% had CD8⁺ T effector memory phenotype, cytokine expression, and secretion profile. No adverse infusional events or dose-limiting toxicities occurred; GVHD developed in 1 patient (grade 2 liver). Ten patients (67%) maintained or achieved response (7 complete response, 1 partial response, 2 stable disease) for at least 3 months after infusion; 4 of the responders had active disease at the time of infusion. With a median follow-up from infusion of 328 days (range, 118-1328 days), median event-free survival and overall survival were 4.9 months (95% confidence interval [CI], 1-19.3 months) and 19.6 months (95% CI, 5.6 months to not reached), respectively. Collection and enrichment of phenotypic CD8⁺ T_M cells is feasible, well tolerated, and associated with a low incidence of GVHD when administered as a manipulated infusion of donor lymphocytes in patients who have relapsed after HCT. This trial was registered at www.clinicaltrials.gov as #NCT01523223.