We have used a standardized 21-day expansion protocol to produce cytokine-induced killer (CIK) cells starting from very small amounts of nucleated cells (approximately 15× 10 6 cells) isolated from cord blood. Mononuclear cells are stimulated with anti CD3 (OKT3) and IFNγ and then expanded with IL-2. Moreover, we show that washouts of cord blood units bags (at the end of the infusion) may be sufficient to yield almost 500× 10 6 CIK by the same expansion protocol. CIK cells show strong cytotoxic activity against a variety of tumor target cell lines including B and T lymphomas and myeloid leukemias. More importantly, expanded cord blood-derived CIK cells are cytotoxic against fresh leukemic blasts and express perforin, granzyme and NKG2D molecule at high levels. The same in vitro protocol has already been used to expand CIK cells from peripheral blood of adult donors under GMP conditions and therefore these observations open up the possibility of imagining a future clinical application of leukemia relapse following cord blood transplantation with CIK cells obtained from the same cord blood unit.