Objective Successful priming and in vitro expansion of anti-leukemia cytotoxic T lymphocytes (CTL) are preliminary conditions for designing approaches of adoptive immunotherapy in patients with hematological malignancies undergoing allogeneic hematopoietic stem cell transplantation (HSCT). In this study, we evaluated the possibility of generating and expanding in vitro CTL directed toward different types of either leukemia or myelodysplastic cells, using both HLA-matched and partially matched donors. Patients and methods Eleven donor/recipient pairs were enrolled; donor-derived dendritic cells, pulsed with patient blast cells, were used to generate CTL. Results Anti-leukemia CTL lines were successfully obtained from 10 of 11 donors. After repeated rounds of stimulation, CTL lines showed, along with an increase in cytotoxic activity, a variable but continuous expansion of cultured cells. In order to increase the magnitude of CTL expansion, two anti-leukemia CTL lines were further stimulated using allogeneic feeder cells, anti-CD3, and low doses of interleukin-2 (IL-2). This stimulation gave rise to 150-fold to 270-fold expansion of the absolute number of cultured cells. Most cultures showed either absent or low reactivity of anti-leukemia CTL against patient non-leukemia cells. Three anti-leukemia CTL lines displayed a more pronounced cytotoxicity against nonmalignant recipient cells, which was always lower than that observed against leukemia blasts (LB). Spectratyping analysis of the TCR-Vβ subfamilies revealed a preferential expansion of oligoclonal populations that persisted in CTL lines following repeated rounds of stimulation. Conclusions Results provide the biological background for designing protocols of adoptive immunotherapy for the control of minimal residual disease in patients with hematological malignancies given HSCT.