Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target in AML. Eradication of LSCs should be a critical and efficient therapeutic approach for the cure of AML. T-cell immunoglobulin mucin-3 (TIM-3) is expressed in most types of AML LSCs, but not in normal hematopoietic stem cells (HSCs); therefore, TIM-3 would be one of the promising therapeutic targets to specifically kill AML LSCs, sparing normal HSCs. In xenograft models reconstituted with human AML LSCs or human normal HSCs, an anti-human TIM-3 mouse antibody with cytotoxic activities exerts a potent anti-leukemic effect by targeting AML LSCs but does not affect normal human hematopoiesis in vivo. Here, we would like to introduce the recent studies on TIM-3 in normal and malignant hematopoiesis.