Acute Myeloid Leukemia (AML) is a malignant disease of the blood which is fatal in over half of adults diagnosed with the disease. It is hypothesized that dysregulated growth signaling may contribute to the pathogenesis of AML but the mechanism of growth dysregulation is poorly understood. Signal transducer and activator of transcription 5 (Stat5) is one such signaling molecule, a transcription factor that regulates many aspects of cell growth, survival and differentiation. Stat5 protein has two different forms, STAT5A and STAT5B, which are encoded by two distinct, yet closely related genes. Constitutive activation of Stat5 has been identified in a number of hematopoietic malignancies including AML. However, it is unclear whether Stat5 activation in AML suggests the activation of STAT5A or STAT5B or both. In this study, activation status of both STAT5A and STAT5B proteins are examined respectively in AML cell lines and primary patient samples, by using co-immunoprecipitation with anti-STAT5A, STAT5B, and p-Stat5 (Tyr694/699) antibodies. Results show that all AML cells contain constitutively phosphorylated STAT5A except one line as well as constitutively phosphorylated STAT5B. To further determine the functional significance of the constitutive activation of STAT5A and STAT5B, knockdown strategy was employed to deplete STAT5A and STAT5B by using siRNA. Depleted expression of STAT5A/B in AML resulted in decreased survival and growth in liquid culture assays and colony forming cells (CFCs) assay. These results demonstrate that both STAT5A and STAT5B are required for AML cell growth and survival. Understanding the role of STAT5A/B should lead to novel therapies for AML and, likely, other malignancies that also contain constitutive activation of STAT5A/B.