Relapse remains the leading cause of mortality in acute myeloid leukemia (AML), largely due to the persistence of therapy-resistant leukemia stem cells (LSCs). However, surface determinants that sustain LSC function and disease aggressiveness remain incompletely defined. Here, we identify the tetraspanin CD81 as a regulator of LSC function, progression and treatment resistance in AML. Analysis of retrospective patient cohorts revealed that high CD81 surface expression is associated with relapse and adverse clinical outcomes in non-core-binding factor AML. Functional studies demonstrated that elevated CD81 expression promotes chemoresistance and enhances leukemic engraftment in immunodeficient mouse models. In vivo gain- and loss-of-function approaches further established that CD81 drives increased leukemia burden and aggressive disease behavior. Notably, CD81 was enriched within LSC-containing subpopulations, where its expression supported LSC maintenance and resistance to chemotherapy. Mechanistically, CD81 promotes chemoresistance and leukemic aggressiveness through pathways linked to LAPTM4B-mediated STAT3 signaling and enhanced adhesion-dependent cellular interactions. These effects were accompanied by increased migration, invasion, and formation of filopodia-like membrane protrusions. Importantly, therapeutic immunotargeting of CD81 significantly reduced leukemic burden while exhibiting a manageable toxicity profile in preclinical models. Collectively, these findings establish CD81 as a clinically relevant surface marker associated with AML relapse and identify CD81-dependent signaling as a therapeutic vulnerability for targeting LSCs and preventing disease recurrence.