Arsenic trioxide (As(2)O(3)) has potent antileukemic properties in vitro and in vivo, but the mechanisms by which it generates its effects on target leukemic cells are not well understood. Understanding cellular mechanisms and pathways that are activated in leukemic cells to control the generation of As(2)O(3) responses should have important implications in the development of novel approaches using As(2)O(3) for the treatment of leukemias. In this study, we used immunoblotting and immune complex kinase assays to provide evidence that the kinases thousand-and-one amino acid kinase 2 (TAO2) and transforming growth factor-beta-activated kinase 1 (TAK1) are rapidly activated in response to treatment of acute leukemia cells with As(2)O(3). Such activation occurs after the generation of reactive oxygen species and regulates downstream engagement of the p38 mitogen-activated protein kinase. Our studies demonstrate that siRNA-mediated knockdown of TAO2 or TAK1 or pharmacological inhibition of TAK1 enhances the suppressive effects of As(2)O(3) on KT-1-derived leukemic progenitor colony formation and on primary leukemic progenitors from patients with acute myelogenous leukemia. These results indicate key negative-feedback regulatory roles for these kinases in the generation of the antileukemic effects of As(2)O(3). Thus, molecular or pharmacological targeting of these kinases may provide a novel approach to enhance the generation of arsenic-dependent antileukemic responses.