Acute myeloid leukemia (AML), an aggressive malignancy with unmet medical need, lacks immunotherapeutic options. CD123, the cellular receptor for interleukin-3, expressed in AML is an attractive target for tumor specific therapy. Vibecotamab (XmAb14045), a humanized bispecific antibody, monovalently binds both CD3 and CD123 to recruit cytotoxic T cells to kill CD123+ tumor cells. This Phase 1 study's primary objectives were safety and tolerability and identification of a maximum tolerated dose/recommended dose for use as monotherapy in patients with relapsed/refractory AML. Identification of a recommended Phase 2 vibecotamab dose comprised 3 step-up doses (Week 1), which were noted to reduce cytokine response syndrome (CRS), followed by weekly dosing (1.7 µg/kg, Cohort -1D). In 16/120 patients, at least 1 treatment-emergent adverse event was classified as a dose-limiting toxicity. CRS, the most common adverse event (59.2%), managed with premedication, were mostly ≤Grade 2. A secondary objective was assessment of efficacy in patients with CD123 expressing leukemias. A total of 10/111 (9.0%) efficacy-evaluable AML patients achieved an overall response of morphologic leukemia free state or better with an overall objective response rate (ORR) of 9.0%. Response was only observed in patients receiving a target dose of 0.75 µg/kg or higher (n = 87) in which the efficacy-evaluable ORR was 11.5%. Response was associated with lower baseline blast count in blood and bone marrow (<25%) suggesting potential benefit. This trial was registered at www.clinicaltrials.gov as #NCT02730312.