Despite high remission rates after therapy, 60% to 70% of patients with acute myeloid leukemia (AML) do not survive 5 years after their initial diagnosis. The main cause of treatment failures may be insufficient eradication of a subpopulation of leukemic stem-like cells (LSC), which are thought to be responsible for relapse by giving rise to more differentiated leukemic progenitors (LP). To address the need for therapeutic targets in LSCs, we compared microRNA (miRNA) expression patterns in highly enriched healthy CD34(+)CD38(-) hematopoietic stem cells (HSC), CD34(+)CD38(-) LSCs, and CD34(+)CD38(+) LPs, all derived from the same patients' bone marrow (BM) specimens. In this manner, we identified multiple differentially expressed miRNAs, in particular miR-126, which was highly expressed in HSCs and increased in LSCs compared with LPs, consistent with a stem-like cell function. High miR-126 expression in AML was associated with poor survival, higher chance of relapse, and expression of genes present in LSC/HSC signatures. Notably, attenuating miR-126 expression in AML cells reduced in vitro cell growth by inducing apoptosis, but did not affect the survival of normal BM in which it instead enhanced expansion of HSCs. Furthermore, targeting miR-126 in LSCs and LPs reduced their clonogenic capacity and eliminated leukemic cells, again in the absence of similar inhibitory effects on normal BM cells. Our results define miR-126 as a therapeutic focus to specifically eradicate LSCs and improve AML outcome.