Induction treatments for acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years and AML remains a disease of poor prognosis. Allogeneic hematopoietic cell transplantation can achieve cures in selected patients and highlights the susceptibility of AML to donor derived immunotherapy. The interleukin 3 receptor alpha chain (CD123) has been identified as a potiential immunotherapeutic target since it is over-expressed on AML compared to normal hematopoietic stem cells. Therefore, we developed two chimeric antigen receptors (CARs) containing a CD123 specific ScFv in combination with a CD28 co-stimulatory domain and CD3-zeta signaling domain, targeting different epitopes on CD123. CD123 CAR redirected T cells mediated potent effector activity against CD123(+) cell lines as well as primary AML patient samples. CD123 CAR T cells did not eliminate granulocyte/macrophage and erythroid colony formation in vitro. Additionally, T cells obtained from patients with active AML can be modified to express CD123 CARs and are able to lyse autologous AML blasts in vitro. Finally, CD123 CAR T cells exhibited anti-leukemic activity in vivo against a xenogenic model of disseminated AML. These results suggest that CD123 CAR T cells are a promising immunotherapy for the treatment of high risk AML.