Evidences obtained in our laboratory indicated that the leukaemia stem cell (LSC) potential of chronic myeloid leukaemia (CML) cell populations is resistant to, and selected in, severe hypoxia (0.1-1% oxygen). The MAPK ERK5 is involved in the control of cell survival and proliferation and in the pathogenesis of different types of cancer, including CML.The main target of this study was to address the effects of ERK5 inhibition on the maintenance of hypoxia-selected LSC of CML by the Culture-Repopulating Ability (CRA) assay. The K562 and KCL22 human CML cell lines, where ERK5 is constitutively activated, were used. Cells were incubated in hypoxic (~0.1% O2) or normoxic (control) primary cultures (LC1) in the absence or the presence of MEK5 or ERK5 inhibitors for different times. Survival, cycling, proliferation and apoptosis were assessed by counting Trypan blue-negative cells, flow cytometry and western blotting. At day 7 of incubation, LC1 cells were transferred to non-selective normoxic secondary cultures (LC2) of CRA assays in the absence of inhibitors, to evaluate LC2 kinetics of repopulation. Hypoxia prevents the cell number increase which occurred in normoxia and determined early and massive apoptosis, as well as cell cycle arrest of surviving cells. Hypoxia decreased the intensity and duration of ERK1/2, p38 and JNK phosphorylation/activation, but not expression, occurring in normoxia. Hypoxia suppressed ERK5 constitutive activation and protein, but not mRNA, expression. We tested several MEK5>ERK5 pathway inhibitors and identified one specific ERK5 inhibitor that completely suppressed LSC maintenance in hypoxia. In LC1, this inhibition resulted in a significantincrease ofthe percentage of cells in G0/G1 and in a modest reduction of apoptosis, pointing to a cytostatic, rather than cytotoxic, effect of ERK5 inhibition. The maintenance of leukaemia stem cells of CML is impaired by ERK5 inhibition, which therefore emerges as a potential novel strategy for CML therapy.