While the emergence of WT1-specific cytotoxic T lymphocytes (WT1-CTL) has been correlated with better relapse-free survival after allogeneic stem cell transplantation in patients with myeloid leukemias, little is known about the role of these cells in multiple myeloma (MM). We examined the significance of WT1-CTL responses in patients with relapsed MM and high-risk cytogenetics who were undergoing allogeneic T cell–depleted hematopoietic stem cell transplantation (alloTCD-HSCT) followed by donor lymphocyte infusions. Of 24 patients evaluated, all exhibited WT1-CTL responses before allogeneic transplantation. These T-cell frequencies were universally correlated with pretransplantation disease load. Ten patients received low-dose donor lymphocyte infusions beginning 5 months after transplantation. All patients subsequently developed increments of WT1-CTL frequencies that were associated with reduction in specific myeloma markers, in the absence of graft-versus-host disease. Immunohistochemical analyses of WT1 and CD138 in bone marrow specimens demonstrated consistent coexpression within malignant plasma cells. WT1 expression in the bone marrow correlated with disease outcome. Our results suggest an association between the emergence of WT1-CTL and graft-versus-myeloma effect in patients treated for relapsed MM after alloTCD-HSCT and donor lymphocyte infusions, supporting the development of adoptive immunotherapeutic approaches using WT1-CTL in the treatment of MM (registered at http://clinicaltrials.gov, ID: NCT01131169).