Although overexpression/activation of focal adhesion kinase (FAK) is widely known in solid tumors to control cell growth, survival, invasion, metastasis, gene expression, and stem cell self-renewal, its expression and function in myeloid leukemia are not well investigated. Using reverse-phase protein arrays in large cohorts of newly diagnosed acute myeloid leukemia (AML) and myeloid dysplastic syndrome (MDS) samples, we found that high FAK expression was associated with unfavorable cytogenetics (P = 2 x 10-4) and relapse (P = 0.02) in AML. FAK expression was significantly lower in patients with FLT3-ITD (P = 0.0024) or RAS (P = 0.05) mutations and strongly correlated with p-SRC and integrinβ3 levels. FAK protein levels were significantly higher in CD34+ (P = 5.42 x 10-20) and CD34+ CD38- MDS (P = 7.62 x 10-9) cells compared to normal CD34+ cells. MDS patients with higher FAK in CD34+ cells tended to have better OS (P = 0.05). FAK expression was significantly higher in MDS patients who later transformed to compared with not transformed to AML and in AML patients who transformed from MDS compared with those with de novo AML. Co-culture with mesenchymal stromal cells (MSCs) increased FAK expression in AML cells. Inhibition of FAK decreased MSC-mediated adhesion/migration and viability of AML cells and prolonged survival in an AML xenograft murine model. Our results suggest that FAK regulates leukemia-stromal interactions and supports leukemia cell survival; hence FAK is a potential therapeutic target in myeloid leukemia.