The molecular mechanisms that regulate DC differentiation and subset distribution are largely unknown. In this study we report the identification of the C(2)H(2) zinc finger transcription factors (TF) CTCF as a regulator of DC differentiation. CTCF was expressed in human and murine DC and its expression was downregulated during the differentiation of human monocyte-derived DC. Enforced expression of CTCF during the differentiation of murine BM-derived DC (BMDC) caused increased apoptosis and reduced proliferation leading to a dramatically reduced number of CTCF transduced DC. The CTCF expressing BMDC that developed had a more immature phenotype than control cells, and showed defects in maturation upon TLR stimulation. Furthermore, in vivo expression of CTCF led to an increase in the percentage of plasmacytoid DC (pDC) within the DC lineage. Our data provide new insight into molecular mechanisms regulating DC differentiation and subset development and identify CTCF as a factor involved in the regulation of these important processes.