HER2-directed therapies, such as trastuzumab and lapatinib, are important treatments for breast cancer. However, some tumors do not respond or develop resistance to these agents. We isolated and characterized multiple lapatinib-resistant, HER2-positive, estrogen receptor (ER)–positive breast cancer clones derived from lapatinib-sensitive BT474 cells by chronic exposure to lapatinib. We show overexpression of AXL as a novel mechanism of acquired resistance to HER2-targeted agents in these models. GSK1363089 (foretinib), a multikinase inhibitor of AXL, MET, and vascular endothelial growth factor receptor currently in phase II clinical trials, restores lapatinib and trastuzumab sensitivity in these resistant cells that exhibit increased AXL expression. Furthermore, small interfering RNA to AXL, estrogen deprivation, or fulvestrant, an ER antagonist, decreases AXL expression and restores sensitivity to lapatinib in these cells. Taken together, these data provide scientific evidence to assess the expression of AXL in HER2-positive, ER-positive patients who have progressed on either lapatinib or trastuzumab and to test the combination of HER2-targeted agents and GSK1363089 in the clinic. [Cancer Res 2009;69(17):6871–8]