Human urothelial cell carcinoma evolves via the accumulation of numerous genetic alterations, with loss of p53 and p16 function representing important stages in the development of superficial lesions and their progression to malignant disease. To investigate the effects of disabling either or both proteins in otherwise normal human urothelial cells, we performed retroviral transductions with a dominant negative p53 miniprotein and/or mutant cyclin-dependent kinase 4 (CDK4R24C) in 3 independent cell lines. Although cells with disabled p53 function showed a higher proliferation rate, inactivation of neither p53 nor p16 function resulted in any extension of life span and the double-transductants failed to flourish, demonstrating that further genetic alterations are required to attain an immortalised phenotype. However, CDK4R24C transductants showed a marked increase in apoptotic susceptibility to membrane-presented CD40 ligand, being intermediate between normal cells (nonsusceptible) and transformed cells (highly susceptible). By contrast, loss of p53 function alone only slightly increased the apoptotic susceptibility of urothelial cells. These results demonstrate that loss of p16 function, while insufficient to immortalise urothelial cells, nevertheless renders the cells more vulnerable to apoptosis induced by CD40 ligation.