Human viruses have traditionally been studied in immortalized, usually tumor-derived, cell lines because of their availability, low cost, ease of expansion, and ease of care. Although most viruses infect, replicate, and spread in these cells, all aspects of in vivo virus-host interaction are not accurately reflected in immortalized cells. The isolation, storage, and differentiation of human airway epithelial basal cells have enabled ex vivo studies of RSV infection in a near natural setting. Here we provide our rationale for using well-differentiated primary human bronchial and nasal epithelial cultures, and a current protocol for generating these fully differentiated airway cultures from donor tissue for ex vivo studies of RSV and other respiratory virus infections.