Acute lymphoblastic leukemia (ALL) is one of the most prevelant pediatric malignancies. Although cure rates have improved in recent decades, approximately one in five children relapse, and survival rates post-relapse remain low. Therefore, more effective and innovative therapeutic strategies are needed in order to improve the outcome in these children. Aurora kinases, a family of serine/threonine kinases essential for regulated mitosis, are overexpressed in many forms of cancer, and have been identified as potential targets for cancer therapeutics. Based on this premise, we evaluated the activity of the Aurora-A/B inhibitor AT9283 against pediatric leukemia cells. It was found that AT9283 significantly inhibited the growth and survival of cell lines derived from patients with pediatric leukemia. Specifically, AT9283 promoted Flt-3 dephosphorylation, inhibiting the activity of downstream effectors such as Erk and Mek. In addition, apoptotic markers were also identified, providing a panel of markers for biological correlative analysis for drug activity. Lastly, drug combination studies demonstrated the potential of several novel and conventional agents to synergize with AT9283, including apicidin, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and doxorubicin. These data provide a rationale for further studies and the formulation of a clinical trial of AT9283 for the treatment of refractory pediatric ALL.