Purpose: Quiescent leukemia stem cells (LSC) are important resources of resistance and relapse in chronic myelogenous leukemia (CML). Thus, strategies eradicating CML LSCs are required for cure. In this study, we discovered that AXL tyrosine kinase was selectively overexpressed in primary CML CD34(+) cells. However, the role of AXL and its ligand Gas6 secreted by stromal cells in the regulation of self-renewal capacity of LSCs has not been well investigated. Experimental Design: The function of CML CD34(+) cells was evaluated by flow cytometer, CFC/replating, long-term culture-initiating cells (LTC-IC), CML mouse model driven by human BCR-ABL gene and NOD-scid-IL2Rg(-/-) (NSI) mice. Results: AXL was selectively overexpressed in primary CML CD34(+) cells. AXL knockdown reduced the survival and self-renewal capacity of human CML CD34(+) cells. Pharmacologic inhibition of AXL reduced the survival and self-renewal capacity of human CML LSCs in vitro and in long-term grafts in NSI mice. Human CML CD34(+) cells conscripted bone marrow-derived stromal cells (BMDSC) and primary mesenchymal stem cells (MSC) to secrete Gas6 to form a paracrine loop that promoted self-renewal of LSCs. Suppression of AXL by shRNA and inhibitor prolonged survival of CML mice and reduced the growth of LSCs in mice. Gas6/AXL ligation stabilizes beta-catenin in an AKT-dependent fashion in human CML CD34(+) cells. Conclusions: Our findings improve the understanding of LSC regulation and validate Gas6/AXL as a pair of therapeutic targets to eliminate CML LSCs. (C) 2016 AACR.