Cl−channels such as the Ca2+activated Cl−channel TMEM16A and the Cl−permeable phospholipid scramblase TMEM16F may affect the intracellular Cl−concentration ([Cl−]i), which could act as an intracellular signal. Loss of airway expression of TMEM16A induced a massive expansion of the secretory cell population like goblet and club cells, causing differentiation into a secretory airway epithelium. Knockout of the Ca2+-activated Cl−channel TMEM16A or the phospholipid scramblase TMEM16F leads to mucus accumulation in intestinal goblet cells and airway secretory cells. We show that both TMEM16A and TMEM16F support exocytosis and release of exocytic vesicles, respectively. Lack of TMEM16A/F expression therefore causes inhibition of mucus secretion and leads to goblet cell metaplasia. The human basal epithelial cell line BCi-NS1.1 forms a highly differentiated mucociliated airway epithelium when grown in PneumaCult™ media under an air liquid interface. The present data suggest that mucociliary differentiation requires activation of Notch signaling, but not the function of TMEM16A. Taken together, TMEM16A/F are important for exocytosis, mucus secretion and formation of extracellular vesicles (exosomes or ectosomes) but the present data do no not support a functional role of TMEM16A/F in Notch-mediated differentiation of BCi-NS1.1 cells towards a secretory epithelium.