To better understand the pathobiology of human congenital bladder abnormalities and disorders associated with dedifferentiation, such as bladder cancer, we must first unravel the biology of normal bladder development. Therefore, we performed microarray analysis focusing on determining the gene expression profile at the initiation of bladder development.RNA was extracted from embryonic day 13 and 18 mouse bladders (anatomically equivalent to 7 and 13 weeks of human gestation) and gene expression was evaluated using microarrays. Alterations in select genes of biological interest were confirmed using real-time quantitative polymerase chain reaction and localization was determined by immunohistochemistry.The genetic profile in the initiating mouse bladder at embryonic day 13 was dominated by transcription factors, retinoic acid signaling genes, Eph/ephrin bidirectional signaling molecules and genes associated with regulating cell cycle and differentiation. Later in development at embryonic day 18 genes associated with smooth muscle, innervation and epithelial differentiation were up-regulated. In addition, we examined the functional role of midkine, which was highly expressed at embryonic day 13, using organ culture and to our knowledge we provide the first evidence that this growth factor up-regulates molecules associated with bladder smooth muscle differentiation.These data provide novel insights into molecules that orchestrate bladder development and highlight genes that may be involved in diseases associated with abnormal differentiation.