Embryonic stem (ES) cells proliferate infinitely while maintaining pluripotency. The POU family transcription factor Oct3/4 is specifically expressed in ES cells and early embryos and plays a critical role in self-renewal of ES cells. However, only a few examples of Oct3/4 target genes have been identified. In this chapter, we describe our strategy to isolate novel Oct3/4 target genes. We first identify genes that are specifically expressed in ES cells by means of digital differential display of expressed sequence tag databases. Reporter gene and gel mobility shift assays are used to confirm the role of Oct3/4. Identification of novel Oct3/4 targets will facilitate our understanding of pluripotency.