Internal tandem duplications (ITDs) of fms-like tyrosine kinase 3 (FLT3) receptor play an important role in the pathogenesis of acute myeloid leukemia (AML) and represent an attractive therapeutic target. ABT-869 has demonstrated potent effects in AML cells with FLT3-ITDs. Here, we provide further evidence that ABT-869 treatment significantly downregulates cyclins D and E but increases the expression of p21 and p27. ABT-869 induces apoptosis through downregulation of Bcl-xL and upregulation of BAK, BID and BAD. We also evaluate the combinations of ABT-869 and chemotherapy. ABT-869 demonstrates significant sequence-dependent synergism with cytarabine and doxorubicin in cell lines and primary leukemia samples. The optimal combination was validated in MV4-11 xenografts. Low-density array analysis revealed the synergistic interaction involved in downregulation of cell cycle and mitogen-activated protein kinase pathway genes. CCND1 and c-Mos were the most significantly inhibited targets on both transcriptional and translational levels. Treatment with short hairpin RNAs targeting either CCND1 or c-Mos further sensitized MV4-11 cells to ABT-869. These findings suggest that specific pathway genes were further targeted by adding chemotherapy and support the rationale of combination therapy. Thus, a clinical trial using sequence-dependent combination therapy with ABT-869 in AML is warranted.