Advances in the treatment of pediatric AML have been modest over the past four decades. Despite maximally intensive therapy, approximately 40% of patients will relapse. Novel targeted therapies are needed to improve outcomes. We identified mesothelin (MSLN), a well-validated target overexpressed in some adult malignancies, to be highly expressed on the leukemic cell surface in a subset of pediatric AML patients. The lack of expression on normal bone marrow cells makes MSLN a viable target for immunotherapies such as T-cell engaging bispecific antibodies (BsAbs) that combine two distinct antibody-variable regions into a single molecule targeting a cancer-specific antigen and the T-cell co-receptor CD3. Using antibody single-chain variable region (scFv) sequences derived from amatuximab-recognizing MSLN, and from either blinatumomab or AMG330 targeting CD3, we engineered and expressed two MSLN/CD3-targeting BsAbs: MSLN-CD3 and MSLN-CD3, respectively. Both BsAbs promoted T-cell activation and reduced leukemic burden in MV4;11:MSLN xenografted mice, but not in those transplanted with MSLN-negative parental MV4;11 cells. MSLN-CD3 induced complete remission in NTPL-146 and DF-5 patient-derived xenograft models. These data validate the in vivo efficacy and specificity of MSLN-targeting BsAbs. Because prior MSLN-directed therapies appeared safe in humans, MSLN-targeting BsAbs could be ideal immunotherapies for MSLN-positive pediatric AML patients.