The superficial dermis of adult human skin contains a complex arcading microvasculature that provides nutrient support to the overlying epidermis. We propose that the unique subpopulations of dermal fibroblasts located in the superficial dermis contribute to the organization and maintenance of this elaborate microvasculature. This possibility was tested in a coculture system in which distinct subpopulations of adult human dermal fibroblasts were grown to form high-density lawns that were then seeded with human umbilical vein vascular endothelial cells (EC). The fibroblast subpopulation cultured specifically from the papillary dermis supported a robust array of highly branched tube-like structures. In contrast, fibroblasts cultured from the reticular dermis provided an anemic level of support for the formation of tube-like structures. These varied interactions with vascular EC were not due to the differential production of the potent pro-angiogenic factors vascular endothelial growth factor-A or fibroblast growth factor-2. Instead, the extracellular matrix and/or molecules bound to this matrix appeared to contain instructions that modulated these differential fibroblast-vascular EC interactions. One matrix-binding growth factor, hepatocyte growth factor/scatter factor, was identified that was both differentially expressed by papillary and reticular dermal fibroblasts and which was shown to be physiologically relevant in the coculture model. These studies highlight the importance of fibroblasts in supporting and maintaining vascular integrity. Furthermore, these studies have important implications for wound repair and may help to explain how fibroblasts contribute to the etiology of nonhealing wounds.