Tissue-engineering and regenerative medicine strategies for the bladder and urinary tract are dependent on the ability to generate adequate numbers of differentiation-competent uro-epithelial cells. In situ, urothelium is a mitotically quiescent, but highly regenerative epithelium. Although evidence supports a resident, basally located urothelial progenitor population, no specific stem cell has been identified. Our aim was to isolate basal and suprabasal urothelial subpopulations and characterize their regenerative and differentiation potentials in vitro. We showed that the low-affinity nerve growth factor receptor (NGFR) is a cell surface-expressed marker that is restricted to basal cells in normal human and porcine urothelia in situ. We used NGFR immunoseparation and differential adherence to collagen to isolate subpopulations of urothelial cells for culture. Isolated basal-derived porcine NGFR⁺ urothelial cells initially showed a higher proliferative and clonogenic phenotype than their suprabasal NGFR⁻ counterparts in vitro. However, after a short period of adaptation to culture, both NGFR⁺ and NGFR⁻ subpopulations became indistinguishable and displayed similar long-term growth and differentiation potentials. Both populations generated hierarchically organized, differentiated tissue equivalents similar to native urothelium, including a fully reconstituted NGFR⁺ basal cell layer by the NGFR⁻ suprabasal-derived population. Similarly, slow collagen-adherent human urothelial cells initially displayed a longer lag phase than rapid-adherent cultures, but after adaptation, both populations showed similar long-term proliferation, exponential growth rates, and capacity to form a functional barrier urothelium. Our results support a model where urothelial cell phenotype is plastic and determined by the niche or local environment. This has direct implications for tissue-engineering strategies requiring urothelial cell expansion and provides a new perspective toward understanding urothelial regeneration and differentiated tissue hierarchy.