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Despite an effective vaccine, measles virus (MeV) remains a critical health burden worldwide. Many fundamental questions remain about its unique biology. Because MeV is a human specific pathogen, research has largely relied on immortalized human cell lines with confirmatory studies in non-human primates or humanized mice. A critical challenge for MeV research is identification of representative in vitro model systems. To study infection, spread, and release in the airways, we utilize primary cultures of airway epithelia cells collected from human donor lungs. This cell culture model recapitulates the pseudostratified columnar epithelial structure and multiple cell types of the conducting airways. In this model, direct cell-to-cell spread of MeV results in well-defined foci termed infectious centers. Here, we demonstrate that infectious center formation following basolateral MeV infection is consistent between two primate species (human and macaque), between two university core facilities that provide primary human airway cultures, and between human primary cells and fresh human tracheal explants. Canine distemper virus infection of differentiated primary cultures of ferret airway epithelial cells also results in infectious center formation suggesting that this mechanism of infection and spread is shared between morbilliviruses. Importantly, the well-defined morphology of the pseudostratified columnar epithelial layer is lost when fetal bovine serum is included in the culture media or when primary cells are passaged twice. Both conditions lead to apical infection by MeV and loss of defined infectious centers. These results suggest that MeV-induced infectious center formation depends on appropriate differentiation, maintenance, and morphology of primary cultures. IMPORTANCE For many years, measles virus (MeV) was assumed to first enter the host via the apical surface of airway epithelial cells and subsequently spread systemically. We and others reported that MeV has an overwhelming preference for entry at the basolateral surface of airway epithelial cells, which led to a fundamental new understanding of how MeV enters a human host. This unexpected observation using well-differentiated primary cultures of airway epithelia from human donors contradicted previous studies using immortalized cultured cells. Here, we show that appropriate differentiation and cell morphology of primary human airway epithelial cells are critical to recapitulate MeV infection patterns and pathogenesis of the in vivo airways. By simply culturing primary cells in media containing serum or passaging primary cultures, erroneous results quickly emerge. These results have broad implications for data interpretation related to respiratory virus infection, spread, and release from human airway epithelial cells. [ABSTRACT FROM AUTHOR]