Abstract The prognosis of acute myeloid leukemia (AML) remains poor in part due to the leukemic bone marrow microenvironment. Our lab has found that CXCL12, a chemokine abundant within the leukemic bone marrow microenvironment, induces apoptosis of AML cells expressing CXCR4, the receptor for CXCL12. However, this CXCL12/CXCR4-induced apoptosis is inhibited by differentiating osteoblasts, which protect AML cells from apoptosis in the bone marrow. Tipifarnib is a farnesyltransferase inhibitor shown to increase progression-free survival in AML patients that express high levels of CXCL12. Here, we report that tipifarnib inhibits the CXCL12/CXCR4-directed migration of AML cells via an ERK independent pathway. Furthermore, tipifarnib enhances CXCL12/CXCR4-mediated AML cell apoptosis via a mechanism that alters expression of apoptosis-regulating proteins. In addition, tipifarnib disrupts AML protection by osteoblasts, increasing AML cell apoptosis. Tipifarnib inhibits the osteoblast-mediated protection of AML cells via disrupting COL1A1 and TNAP, proteins essential for extracellular matrix production. In conclusion, tipifarnib alters the bone marrow microenvironment which is predicted to enhance eradication of AML via inhibiting CXCL12/CXCR4 directed cellular migration of AML cells, reducing the protective effects of differentiating osteoblasts by disrupting matrix protection proteins, and increasing CXCL12/CXCR4-mediated apoptosis.