Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target. The T-cell immunoglobulin mucin-3 (TIM-3) is expressed on the surface of LSCs in most AML patients. We have reported that targeting TIM-3 by anti-TIM-3 monoclonal antibodies could eradicate human AML LSCs in vivo by utilizing xenograft models (Cell Stem Cell, 2011). We then tested the role of TIM-3 signaling evoked by its ligand, galectin-9 (Gal-9), and found that TIM-3+ AML cells secreted Gal-9 into sera, and the ligation of TIM-3 by serum galectin-9 positively regulate the self-renewal capacity of TIM-3+ LSCs through activating the beta-catenin pathway (Cell Stem Cell, 2015). Furthermore, this TIM-3/Gal-9 “autocrine stimulatory loop” is involved in development of LSCs from preleukemic status, including myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN); frequencies of TIM-3+ cells progressively increased and accumulate driver mutations along with disease progression from early/chronic phase to overt leukemia. Thus, signaling molecules downstream of TIM-3 as well as surface TIM-3 itself might be good target to regulate transformation from preleukemic status.