Epithelial cells are vital for maintaining the complex architecture and functions of organs in the body. Directed by cues from the extracellular matrix, cells polarize their surface into apical and basolateral domains, and connect by extensive cell-cell junctions to form tightly vowen epithelial layers. In fully polarized cells, primary cilia project from the apical surface. Madin-Darby canine kidney (MDCK) cells provide a model to study organization of cells as monolayers and also in 3D in cysts. In this study retrovirus-mediated RNA interference (RNAi) was used to generate a series of knockdowns (KDs) for proteins implicated in apical transport: annexin-13, caveolin-1, galectin-3, syntaxin-3, syntaxin-2 and VIP17 and/or MAL. Cyst cultures were then employed to study the effects of these KDs on epithelial morphogenesis. Depletion of these proteins by RNAi stalled the development of the apical lumen in cysts and resulted in impaired ciliogenesis. The most severe ciliary defects were observed in annexin-13 and syntaxin-3 KD cysts. Although the phenotypes demonstrate the robustness of the formation of the polarized membrane domains, they indicate the important role of apical membrane biogenesis in epithelial organization.