Deciphering the molecular basis of pluripotency is fundamental to our understanding of development and embryonic stem cell function. Here, we report that TAF3, a gene regulatory factor, is highly enriched in ES cells. In this context, TAF3 is required for endoderm lineage differentiation and prevents premature specification of neuroectoderm and mesoderm. Interactions between TAF3 and regulatory proteins CTCF and cohesin were observed, suggesting that these proteins work together to maintain pluripotency. Together, our findings support a new role of TAF3 in mediating long-range gene regulatory interactions that safeguard the finely-balanced transcriptional programs underlying pluripotency.