Urothelial cells line the urinary tract, including the renal pelvis, ureters, bladder, superior urethra, and the central ducts of the prostate. They are highly specialized epithelial cell types possessing unique features, imparting important functional roles in the urinary system. They act as a permeability barrier and protect underlying muscle tissues from the caustic effects of urine while also expanding with bladder filling to adjust urine pressures. The multilayered urothelium is typically structured with differentiated, mature surface cells and less mature basal cells. The basal cell layer contains tissue-specific stem cells able to self-renew for the lifetime of the mammal and also produces a pool of maturing cells for tissue homeostasis. Maintaining regenerative basal cells in a culture facilitates urothelial cell growth in vitro. Additionally, epithelial–mesenchymal communication, epithelial–matrix interactions, and cytokines/growth factors are required to maintain the normal structure and function of mature urothelial cells in vitro and to induce stem cell differentiation into urothelial cells. These cultures are useful to study the biology and physiology of the urinary tract, particularly for the development of cell-based tissue engineering strategies in urology. This chapter describes methods for the isolation of urothelial cells and their maintenance in monolayer culture, and methods for the production of multilayer urothelial cell sheets and three-dimensional cocultures of urothelial and mesenchymal cells.