Novel approaches to healing of chronic wounds, such as venous leg ulcers, include the use of tissue-engineered skin substitutes, e.g., human fibroblast-derived dermis. The exact mechanisms of action of these products and their effects on wound healing at a cellular level are yet to be fully defined. The aim of our study was to evaluate the potential effects of human fibroblast-derived dermis on the healing of chronic wounds using an experimental model. We used a tissue expansion model to examine the effect of human fibroblast-derived dermis on the growth of human tissue biopsied from venous leg ulcers. Further characterization of the cytokine profile produced by human fibroblast-derived dermis in culture was performed using enzyme-linked immunosorbent assay techniques. Addition of medium conditioned with human fibroblast-derived dermis significantly increased the outgrowth of cells from venous leg ulcer biopsies (p = 0.001). We detected bioactive levels of hepatocyte growth factor/scatter factor and interleukin-8 in media conditioned with human fibroblast-derived dermis. Therefore, conditioned media from human fibroblast-derived dermis enhances ex vivo expansion of tissue taken from chronic venous leg ulcers, and contains potent angiogenic factors. These experimental findings may explain the enhanced healing seen with clinical applications of human fibroblast-derived dermis on chronic wounds.