Updated project metadata. Atmospheric oxygen tension is increasingly recognized to be hyperoxic for the maintenance of in vitro cell cultures. Oxygen has broad implications on energy metabolism, cellular growth and many regulatory functions. Primary airway epithelial cells (AECs) differentiated at air-liquid interface (ALI) are the gold standard for preclinical assessment of cystic fibrosis transmembrane conductance regulatory (CFTR) modulator efficacy in CF. The fidelity of CF AECs cultured at atmospheric oxygen tension rather than at reduced oxygen tension which more closely reflect the in vivo environment has not been studied to date. This study examined the impact of ambient (21%) and low (2%) oxygen tension on the expansion and differentiation of nasal epithelial cells (hNECs) derived from 11 participants (8 with CF and 3 non-CF). hNECs expanded under normoxic and chronic hypoxic conditions demonstrated epithelial cobblestone morphology and similar proliferation rate. hNECs differentiated at hypoxia demonstrated poorer differentiation capacity (significantly thinner epithelium and lower TEER) and a shift from ciliated to secretory epithelial phenotype. Hypoxic differentiated hNECs had significantly shorter cilia length, slower beating frequency but had improved cilia coordination. CFTR functional response is altered in hypoxic differentiated hNECs. This study highlights the need to reconsider the oxygen tension used in CF primary cell cultures so as to preserve the characteristics and functional response of the cell models as we progress towards personalised medicine in CF.