Respiratory syncytial virus (RSV) infection is a primary cause of bronchiolitis and hospitalization in infants and is associated with significant mortality and morbidity. To model RSV infection in infant bronchial epithelium, we developed a robust protocol for bronchial basal stem cell (BSC) derivation from tracheal aspirates (TAs) of intubated neonatal patients. TA BSCs can be expanded while retaining the ability to differentiate into all the major types of bronchial epithelial cells in air-liquid interface (ALI), making them an invaluable source to generate functional bronchial epithelium of infants. This protocol outlines the specific steps in utilizing TA BSCs to model RSV infection of infant bronchial epithelium in ALI culture.