The skin is a unique organ that contains two different subsets of dendritic cells, i.e., Langerhans cells and dermal dendritic cells. Our hypothesis is that cutaneous fibroblasts may affect the development of these dendritic cells. We cocultured cord blood CD34⁺ hematopoietic progenitor cells with several human cutaneous fibroblast cell lines without any exogenous cytokines for 3 wk. In this culture, hematopoietic progenitor cells increased in number from 20.1 ± 2.4 times, and produced aggregates of cells with dendritic processes. They were composed of 54.9 ± 3.2% HLA-DR⁺ CD14⁺ CD1a^– cells and 13.8 ± 3.6% HLA-DR⁺ CD1a⁺ cells, which also expressed CD11b and CD11c. There were significant numbers of factor XIIIa⁺ cells in the culture, whereas no Lag⁺ or E-cadherin⁺ cells were detected, and they were potent stimulators in allogeneic T cell activation. There was a significant difference in the ability to induce CD1a⁺ cells among different human cutaneous fibroblast cell lines. These CD1a⁺ cells lacked the expression of CD80, CD86, or CD83. In addition, half of them still expressed CD14. When these dendritic cells were cultured with tumor necrosis factor-α, however, they became mature dendritic cells with augmented expression of CD86 and CD83 and with increased allogeneic T cell stimulation. The subsequent experiment using a dividing chamber, enzyme-linked immunosorbent assay for granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor, and the blocking studies with antibodies for these cytokines suggested that both the presence of direct contact between hematopoietic progenitor cells and human cutaneous fibroblast cell lines and macrophage colony-stimulating factor produced by human cutaneous fibroblast cell lines are required for their maximum growth and differentiation into CD1a⁺ dendritic cells, whereas macrophage colony-stimulating factor was solely responsible for their differentiation. These data suggest that cutaneous fibroblasts support the differentiation of dermal dendritic cells in addition to that of monocytes from hematopoietic progenitor cells by their direct contact with hematopoietic progenitor cells and by their macrophage colony-stimulating factor production.