The Wilms tumor antigen, WT1, is expressed at high levels in various types of leukemia and solid tumors, including lung, breast, colon cancer and soft tissue sarcomas. The WT1 protein has been found to be highly immunogenic, and spontaneous humoral and cytotoxic T-cell responses have been detected in patients suffering from leukemia. Furthermore, major histocompatibility complexes class I- and II-restricted WT1 peptide epitopes have been shown to elicit immune responses in patients with WT1-expressing tumors. As a consequence, WT1 has become an attractive target for anticancer immunotherapy. In this study, we investigated the feasibility of generating WT1-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation. We analyzed the incidence of T cells specific for WT1 peptide epitopes in cancer patients and healthy volunteers. It is noted that we could generate WT1-specific responses in nine of ten healthy volunteer donors and established T-cell clones specific for two WT1-derived peptide epitopes. These in vitro expanded WT1-specific T cells effectively lysed WT1-expressing tumor cell lines, indicating the potential clinical impact of ex vivo expanded donor-derived WT1-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation.