AbstractVacuole membrane protein (VMP1) is a putative autophagy protein, which together with Beclin-1 acts as a molecular switch in activating autophagy. In the present study the role of VMP1 was analysed in CD34⁺ cells of cord blood (CB) and primary acute myeloid leukemia (AML) cells and cell lines. An increased expression of VMP1 was observed in a subset of AML patients. Functional studies in normal CB CD34⁺ cells indicated that inhibiting VMP1 expression reduced autophagic-flux, coinciding with reduced expansion of hematopoietic stem and progenitor cells (HSPC), delayed differentiation, increased apoptosis and impaired in vivo engraftment. Comparable results were observed in leukemic cell lines and primary AML CD34⁺ cells. Ultrastructural analysis indicated that leukemic cells overexpressing VMP1 displayed a reduced number of mitochondrial structures, while the number of lysosomal degradation structures was increased. The overexpression of VMP1 did not affect cell proliferation and differentiation, but increased autophagic-flux and improved mitochondrial quality, which coincided with an increased threshold for venetoclax-induced loss of mitochondrial outer membrane permeabilization (MOMP) and apoptosis. In conclusion, our data indicate that in leukemic cells high VMP1 is involved with mitochondrial quality control.