The CD3×CD123 DART induces redirected killing of human AML blasts in the presence and absence of stroma. Cells from AML patient 9 were grown in the presence or absence of NSG stroma in media supplemented with IL-2 or a mixture of IL-2 and murine macrophage-colony-stimulating factor (100 ng/mL), murine IL-3, human IL-6, murine thrombopoietin, and human FLT3L. Twelve hours after initiation of culture, PBS (No DART) or 1 ng/mL CD3×CD123 DART were added to the wells. Six days later, cells were harvested and analyzed by flow cytometry for CD45^dimSSC^lo AML blasts expressing CD34 and CD123. (A) Representative flow cytometry of CD34 and CD123 expression on CD45^dimSSC^lo AML blasts. Bar graph represents the percentage of cells of the indicated phenotype out of all blasts in the wells. (B) The absolute numbers of total CD45^dimSSC^lo AML blasts and CD45^dimSSC^lo AML cells expressing CD34 and CD123. Each data point represents the average of 4 separate experiments, where samples were analyzed in duplicate or triplicate in each experiment. The average number of cells under different culture conditions were summarized using means and standard deviations and compared by two-way ANOVA for repeated measurement data. As a result of relatively large variability in data, a logarithm transformation was performed to better satisfy the assumption of normal distribution. All the tests were two-sided, and a P-value of .05 or less was taken to indicate statistical significance. *P < .05, **P < .01, ***P < .001.