Hepatocyte nuclear factor 4 alpha (Hnf4alpha) is a transcription factor required for embryogenesis and organogenesis. In the adult kidney, Hnf4alpha is expressed at a high level in proximal tubules. Although its expression begins from the embryonic period, its function in the developing kidney has remained almost unknown. In this study, we investigated the role of Hnf4alpha in the cultured embryonic mouse kidney by gene silencing using the RNA interference method. Additionally, we identified the dynamics of Hnf4alpha in the microenvironment of the developing kidney. As a result of gene silencing, the cellular organization in the condensed mesenchyme (CM) fell into disorder and many apoptotic cells appeared. In addition, laser microdissection-reverse transcriptase-polymerase chain reaction provided evidence that Hnf4alpha gene expression began first in the CM. These results suggest the possibility that Hnf4alpha plays an important role in the regulation of the cell survival at the CM stage in nephrogenesis.