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Purpose: The urothelial stroma is presumed to have a critical role in the formation and homeostasis of normal urothelium. To determine the intrinsic capacity of urothelial cells to initiate urothelial differentiation human urothelial cell were cultured under conditions that promote differentiation in the absence of stromal signaling., Materials and Methods: Immortalized and primary human urothelial cells were cultured in semisolid medium. Recovered cells were then analyzed by immunofluorescence, flow cytometry and immunoblotting for expression of the differentiation specific keratins K18 and K8, and cyclin-cyclin-dependent kinase inhibitors. The expression of these markers in cells following semisolid culture was then compared with that in normal bladder and ureteral mucosa as well as in synthetic urothelium generated by 3-dimensional organotypic raft cultures., Results: Organotypic raft culture of primary and immortalized urothelial cells generated full-thickness epithelium that resembled human bladder and ureteral urothelium, and expressed K8 and K18 in superficial layers. Suspension culture in semisolid medium induced K18 expression approximately 9-fold at 24 hours. p21 and p27 expression were induced by 6 hours and yet p21 expression subsided within 12 hours, while p27 expression persisted., Conclusions: These results indicate that primary and immortalized human urothelial cells have the capacity to enter the urothelial differentiation program and such entry does not require inductive signals from stroma. Furthermore, these data suggest that p21 and p27 have distinct roles in regulating the urothelial cell cycle.