Mutations in the metabolic enzymes isocitrate dehydrogenase-1 (IDH1) and IDH2 that produce the oncometabolite D-2-hydroxyglutarate (2-HG) occur frequently in human acute myeloid leukemia (AML). 2-HG modulates numerous biological pathways implicated in malignant transformation, but the contribution of mutant IDH proteins to maintenance and progression of AML in vivo is currently unknown. To answer this crucial question we have generated transgenic mice that express IDH2R140Q in an on/off- and tissue-specific manner using a tetracycline-inducible system. We found that IDH2R140Q can cooperate with overexpression of HoxA9 and Meis1a and with mutations in FMS-like tyrosine kinase 3 (FLT3) to drive acute leukemia in vivo. Critically, we show that genetic deinduction of mutant IDH2 in leukemic cells in vivo has profound effects on their growth and/or maintenance. Our data demonstrate the proto-oncogenic role of mutant IDH2 and support its relevance as a therapeutic target for the treatment of human AML. Display omitted •Development of a doxycycline-inducible mouse model of IDH2R140Q•IDH2R140Q drives aberrant self-renewal and a block of differentiation in HSPCs•IDH2R140Q cooperates with Flt3ITD to drive acute leukemia in vivo•IDH2R140Q is essential for leukemia maintenance Kats et al. show that mutant IDH2 drives self-renewal of HSPCs and cooperates with oncogenes FLT3, HoxA9, and Meis1a to drive initiation of leukemia in vivo.