A substantial number of patients with leukemia and lymphoma treated with anti-CD19 or anti-CD22 monoCAR-T cell therapy relapse because of antigen loss or down-regulation. We hypothesized that B cell tumor antigen escape may be overcome by a chimeric antigen receptor (CAR) design that simultaneously targets three B cell leukemia antigens. We engineered trispecific duoCAR-T cells with lentiviral vectors encoding two CAR open reading frames that target CD19, CD20, and CD22. The duoCARs were composed of a CAR with a tandem CD19- and CD20-targeting binder, linked by the P2A self-cleaving peptide to a second CAR targeting CD22. Multiple combinations of intracellular T cell signaling motifs were evaluated. The most potent duoCAR architectures included those with ICOS, OX40, or CD27 signaling domains rather than those from CD28 or 4-1BB. We identified four optimal binder and signaling combinations that potently rejected xenografted leukemia and lymphoma tumors in vivo. Moreover, in mice bearing a mixture of B cell lymphoma lines composed of parental triple-positive cells, CD19-negative, CD20-negative, and CD22-negative variants, only the trispecific duoCAR-T cells rapidly and efficiently rejected the tumors. Each of the monoCAR-T cells failed to prevent tumor progression. Analysis of intracellular signaling profiles demonstrates that the distinct signaling of the intracellular domains used may contribute to these differential effects. Multispecific duoCAR-T cells are a promising strategy to prevent antigen loss-mediated relapse or the down-regulation of target antigen in patients with B cell malignancies.