Background: The recently identified bladder cancer stem cells are considered to be mediators of resistance to current therapies and therefore represent strong candidate biological targets. To characterize bladder cancer stem cells at the molecular level, the expression of oncoproteins that have been implicated in the self-renewal of adult or embryonic stem cells, is detected. These oncoproteins include Nanog and STAT3. Objectives: The aim of this study was to detect the presence of cancer stem cells of bladder cancer by detection of the two genes, Stat3 and Nanog in cultured bladder tumor biopsies obtained from 15 recently diagnosed subjects with transitional cell carcinoma of the urinary bladder. Tumor tissue was disrupted and cultured in serum free medium. Cancer stem cells were positively selected through serial passaging. RNA extraction and reverse transcription into cDNA was done. Nanog and STAT3 expression was detected through SYBR Green real time PCR. The genes expression levels were compared to their expression in 15 control normal bladder tissue biopsies. Results: Statistical comparison for the expression levels of Nanog and STAT3 between the bladder carcinoma patients cultured tissue and the controls tissue showed a highly statistical significant difference, P value <0.001 for Nanog and P value = 0.003 for STAT3. Conclusion: Nanog and STAT3 genes expression in bladder cancer cultured cells support the conclusion that bladder cancer stem cells are implicated in bladder cancer pathogenesis.