Allogeneic stem cell transplantation (SCT) from cord blood (CB) as a stem cell source is a promising alternative when no human leukocyte antigen-matched donor is found. Donor lymphocyte infusion (DLI) is a possible treatment modality for threatening graft failure or relapse of an underlying malignancy after transplantation. Ethical and logistical reasons limit the possibility of DLI in the setting of CB SCT. To remedy this restriction, we performed expansion of donor T cells in vitro from CB grafts in a clinical setting for use as future DLI and characterized the expanded cells in comparison to T cells from CB acquired ex vivo and adult peripheral blood. T cells were expanded from grafts used for transplantation, upon CD3/CD28 crosslinking and culture in interleukin-2. Phenotype and function of T cells were assessed by flow cytometry and mixed lymphocyte culture assays. T-cell receptor repertoire distribution was evaluated with polymerase chain reaction-based spectratyping. We were able to amplify T cells to sufficient amounts for DLI in 13 out of 13 initiated expansions. Expanded T cells presented with an activated phenotype and could be induced to produce cytokines by a nonspecific stimulus. When exposed to allogeneic targets, expanded CB T cells proliferated at comparable levels to their ex vivo and adult blood counterparts. In summary, clinical expansion of CB T cells for DLI is feasible and may be a future modality for treatment of graft failure or relapse after SCT.