The role of autophagy during leukemia treatment is unclear. On the one hand, autophagy might be induced as a prosurvival response to therapy, thereby reducing treatment efficiency. On the other hand, autophagy may contribute to degradation of fusion oncoproteins, as recently demonstrated for promyelocytic leukemia-retinoic acid receptor α and breakpoint cluster region-abelson, thereby facilitating leukemia treatment. Here, we investigated these opposing roles of autophagy in t(8;21) acute myeloid leukemia (AML) cells, which express the most frequently occurring AML fusion oncoprotein, AML1-eight-twenty-one (ETO). We demonstrate that autophagy is induced by AML1-ETO-targeting drugs, such as the histone deacetylase inhibitors (HDACis) valproic acid (VPA) and vorinostat. Furthermore, we show that autophagy does not mediate degradation of AML1-ETO but rather has a prosurvival role in AML cells, as inhibition of autophagy significantly reduced the viability and colony-forming ability of HDACi-treated AML cells. Combined treatment with HDACis and autophagy inhibitors such as chloroquine (CQ) led to a massive accumulation of ubiquitinated proteins that correlated with increased cell death. Finally, we show that VPA induced autophagy in t(8;21) AML patient cells, and combined treatment with CQ enhanced cell death. Because VPA and CQ are well-tolerated drugs, combinatorial therapy with VPA and CQ could represent an attractive treatment option for AML1-ETO-positive leukemia.