Allogeneic hematopoietic stem-cell transplantation can cure some patients with high-risk B-cell malignancies, but disease relapse following transplantation remains a significant problem. One approach that could be used to augment the donor T-cell–mediated antitumor effect is the infusion of allogeneic donor–derived T cells expressing a chimeric antibody receptor (CAR) specific to the B-cell antigen CD19. However, the use of such cells might result in toxicity in the form of graft-versus-host disease mediated by CD19-specific (CD19-CAR) T cells possessing alloreactive endogenous T-cell receptors. We therefore investigated whether nonalloreactive tumor-specific human T cells could be generated from peripheral blood mononuclear cells of healthy donors by the combination of CD19 redirection via CAR expression and subsequent alloanergization by allostimulation and concomitant blockade of CD28-mediated costimulation. Alloanergization of CD19-CAR T cells resulted in efficient and selective reduction of alloresponses in both CD4⁺ and CD8⁺ T cells, including allospecific proliferation and cytokine secretion. Importantly, T-cell effector functions including CAR-dependent proliferation and specific target cytolysis and cytokine production were retained after alloanergization. Our data support the application of CD19 redirection and subsequent alloanergization to generate allogeneic donor T cells for clinical use possessing increased antitumor activity but limited capacity to mediate graft-versus-host disease. Immunotherapy with such cells could potentially reduce disease relapse after allogeneic transplantation without increasing toxicity, thereby improving the outcome of patients undergoing allogeneic transplantation for high-risk B-cell malignancies. Cancer Res; 70(10); 3915–24. ©2010 AACR.