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Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus with latent and lytic cycles. EBV replicates in the stratified epithelium but the nasopharynx is also composed of pseudostratified epithelium with distinct cell types. Latent infection is associated with nasopharyngeal carcinoma (NPC). Here, we show with nasopharyngeal conditionally reprogrammed cells cultured at the air-liquid interface that pseudostratified epithelial cells are susceptible to EBV infection. Donors varied in susceptibility to de novo EBV infection, but susceptible cultures also displayed differences with respect to pathogenesis. The cultures from one donor yielded lytic infection but cells from two other donors were positive for EBV-encoded EBERs and negative for other lytic infection markers. All cultures stained positive for the pseudostratified markers CK7, MUC5AC, α-tubulin in cilia, and the EBV epithelial cell receptor Ephrin receptor A2. To define EBV transcriptional programs by cell type and to elucidate latent/lytic infection-differential changes, we performed single cell RNA-sequencing on one EBV-infected culture that resulted in alignment with many EBV transcripts. EBV transcripts represented a small portion of the total transcriptome (~0.17%). All cell types in the pseudostratified epithelium had detectable EBV transcripts with suprabasal cells showing the highest number of reads aligning to many EBV genes. Several restriction factors (IRF1, MX1, STAT1, C18orf25) known to limit lytic infection were expressed at lower levels in the lytic subcluster. A third of the differentially-expressed genes in NPC tumors compared to an uninfected pseudostratified ALI culture overlapped with the differentially-expressed genes in the latent subcluster. A third of these commonly perturbed genes were specific to EBV infection and changed in the same direction. Collectively, these findings suggest that the pseudostratified epithelium could harbor EBV infection and that the pseudostratified infection model mirrors many of the transcriptional changes imposed by EBV infection in NPC. Author summary: It has been known for over 50 years that EBV latent infection is associated with NPC. Despite many advances from studies in 2-dimensional cell culture, many aspects of EBV molecular pathogenesis in the nasopharynx remain undefined because the cell types and the biology of the nasopharyngeal epithelium can only be faithfully captured in 3-dimensional cell culture. In the stratified epithelium, cellular differentiation triggers lytic infection but it is not clear to what degree the pseudostratified epithelium is involved. The pseudostratified epithelium is abundant in the lateral wall where the lymphoid-rich fossa of Rosenmüller is located and is a site where NPC tumors most often arises. While the oral epithelium is a site of EBV replication, whether the nasopharyngeal epithelium is a major source of EBV shedding in the nasopharynx is not well defined. Here, we present a 3-dimensional organoid model of the nasopharyngeal pseudostratified epithelium showing that such cells can be infected with EBV in some donor cultures, with examples of both latent and lytic infection. We propose that the cell types of the pseudostratified epithelium should be considered a component of EBV pathogenesis in the nasopharynx and that the difference in donor susceptibility and latent/lytic infection could influence EBV's fitness in the nasopharynx. [ABSTRACT FROM AUTHOR]