Lysine-specific demethylase 1 (KDM1A) is a transcriptional coregulator that can function in both the activation and repression of gene expression, depending upon context. KDM1A plays an important role in hematopoiesis and was identified as a dependency factor in leukemia stem cell populations. Therefore, we investigated the consequences of inhibiting KDM1A in a panel of cell lines representing all acute myelogenous leukemia (AML) subtypes using selective, reversible and irreversible KDM1A small-molecule inhibitors. Cell models of AML, CML, and T-ALL were potently affected by KDM1A inhibition, and cells bearing RUNX1-RUNX1T1 (AML1-ETO) translocations were especially among the most sensitive. RNAi-mediated silencing of KDM1A also effectively suppressed growth of RUNX1-RUNX1T1-containing cell lines. Furthermore, pharmacologic inhibition of KDM1A resulted in complete abrogation of tumor growth in an AML xenograft model harboring RUNX1-RUNX1T1 translocations. We unexpectedly found that KDM1A-targeting compounds not only inhibited the catalytic activity of the enzyme, but evicted KDM1A from target genes. Accordingly, compound-mediated KDM1A eviction was associated with elevated levels of local histone H3 lysine 4 dimethylation, and increased target gene expression, which was further accompanied by cellular differentiation and induction of cell death. Finally, our finding that KDM1A inhibitors effectively synergize with multiple conventional as well as candidate anti-AML agents affords a framework for potential future clinical application. Cancer Res; 76(7); 1975-88. ©2016 AACR.