The critical importance of plasmacytoid dendritic cells (pDCs) in viral infection, autoimmunity, and tolerance has focused major attention on these cells that are rare in blood and immune organs of humans and mice. The recent development of an Flt-3 ligand (FL) culture system of bone marrow cells has led to the simple generation of large numbers of pDCs that resemble their in vivo steady-state counterparts. The FL system has allowed unforeseen insight into the biology of pDCs, and it is assumed that FL is the crucial growth factor for these cells. Surprisingly we have found that a cell type with high capacity for interferon-alpha (IFN-alpha) production in response to CpG-containing oligonucleotides, a feature of pDCs, develop within macrophage-colony-stimulating factor (M-CSF)-generated bone marrow cultures. Analysis of this phenomenon revealed that M-CSF is able to drive pDCs as well as conventional DCs (cDCs) from BM precursor cells in vitro. Furthermore, application of M-CSF to mice was able to drive pDCs and cDCs development in vivo. It is noteworthy that using mice deficient in FL indicated that the M-CSF-driven generation of pDCs and cDCs in vitro and in vivo was independent of endogenous FL.