High BDCA3 expression is associated with a specific human IFN--producing dendritic cell (DC) subset. However, BDCA3 has also been detected on other DC subsets. Thus far, development and function of BDCA3 expression on DCs remains poorly understood. Human Langerhans cells (LCs) and interstitial DCs (intDCs) can be generated in vitro by differentiation of CD34(+) hematopoietic progenitors via distinct precursor DCs (preDCs), CD1a(+) preDCs, and CD14(+) preDCs, respectively. Here, we identified BDCA3 expression in this well-known GM-CSF/TNF--driven culture system and described the effect of IL-4 and/or TGF- on induction of BDCA3 expression. In control or TGF- cultures, BDCA3 was only detected on CD14(+) preDC-derived intDCs. IL-4 induced BDCA3 expression in both CD14(+)-derived and CD1a(+)-derived cultures. TGF- and IL-4 together further increased CD14(+)-derived and CD1a(+)-derived BDCA3(+) DC frequencies, which partly expressed CLEC9A, but were not identical to the BDCA3(high)CLEC9A(+) DC subset in vivo. Importantly, BDCA3(+) cells, but not BDCA3(-) cells, in this system produced high IFN- levels upon polyinosinic:polycytidylic acid (polyI:C) stimulation. This culture system, in which BDCA3 expression is preferentially associated with the intDC lineage and IFN--producing capacity, will greatly contribute to further research on the function and regulation of BDCA3 expression and IFN- production by DCs.