N7-methylguanosine (m7G), a prevalent modification in tRNAs, is primarily catalyzed by the methyltransferase METTL1. While growing evidence supports a role for METTL1 in various tumors, its therapeutic potential and precise function in leukemia stem cell (LSC) homeostasis remain largely unexplored. Here, we identify METTL1 as a key regulator of LSC self-renewal and homing within bone marrow (BM) microenvironment through catalyzing m7G formation on a specific tRNA, tRNAPheGAA, thereby driving leukemogenesis. Mechanistically, METTL1 loss significantly reduces m7G abundance and steady-state levels of tRNAPheGAA, leading to translation suppression and degradation of transcripts enriched with tRNAPheGAA-related codons, such as tyrosine-protein kinase HCK. Decreased HCK expression disrupts CXCR4 signaling, impairing LSC self-renewal and BM homing. Therapeutically, we characterize a small-molecule METTL1 inhibitor (M1i; NSC137443), through high throughput screening. Pharmacological inhibition of METTL1 demonstrates potent anti-tumor efficacy by reduction of tRNA m7G levels and disrupting the tRNAPheGAA/HCK/CXCR4 cascade. Notably, targeting METTL1 significantly reduces LSC frequency, delays leukemogenesis, and prolongs survival in multiple acute myeloid leukemia models. Our findings establish a previously unrecognized role for METTL1 and its target tRNAPheGAA in LSC homeostasis and provide compelling proof-of-concept evidence that METTL1 is a druggable epitranscriptomic target for anti-leukemia therapy.