Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1E912A mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28B upregulation. A small-molecule tool compound antagonizes ADAR1’s effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling. Display omitted •JAK2 signaling activates ADAR1-mediated A-to-I RNA editing•JAK2 and BCR-ABL1 signaling converge on ADAR1 activation through STAT5a•ADAR1-mediated microRNA editing impairs let-7 biogenesis and enhances LSC self-renewal•JAK2 and BCR-ABL1 inhibition reduces ADAR1 expression and prevents LSC self-renewal Zipeto, Court, and colleagues show a pivotal role for let-7 microRNA editing in leukemia stem cell self-renewal. Impairment of let-7 is dependent on JAK2 and BCR-ABL-mediated activation of ADAR1 editing. This provides a novel mechanism of malignant reprogramming that can be targeted through combined JAK2 and BCR-ABL or ADAR1 inhibition.