Antibody-based immunotherapy represents a promising strategy to target and eliminate chemoresistant leukemic cells. Here, we evaluated the CD33/CD3-bispecific T cell engaging (BiTE) antibody (AMG 330) for its suitability as a therapeutic agent in acute myeloid leukemia (AML). We first assessed CD33 expression levels by flow cytometry and found expression in >99% of patient samples (n = 621). CD33 was highest expressed in AMLs with NPM1 mutations (P < .001) and lower in AMLs with complex karyotypes and t(8;21) translocations (P < .001). Furthermore, leukemic stem cells within the CD34⁺/CD38⁻ compartment displayed CD33 at higher levels than healthy donor stem cells (P = .047). In MS-5 feeder cell-based long-term cultures that supported the growth of primary AML blasts for up to 36 days, AMG 330 efficiently recruited and expanded residual CD3⁺/CD45RA⁻/CCR7⁺ memory T cells within the patient sample. Even at low effector to target ratios, the recruited T cells lysed autologous blasts completely in the majority of samples and substantially in the remaining samples in a time-dependent manner. This study provides the first correlation of CD33 expression levels with AML genotype in a comprehensive analysis of adult patients. Targeting CD33 ex vivo using AMG 330 in primary AML samples led to T cell recruitment and expansion and remarkable antibody-mediated cytotoxicity, suggesting efficient therapeutic potential in vivo.