There is accumulating evidence that interactions between epidermal melanocytes and stromal cells play an important role in the regulation of skin pigmentation. In this study we established a pigmented dermo-epidermal skin model (melDESS) of human origin to investigate the effects of distinct stromal cells on melanogenesis. melDESS is a complex, clinically relevant skin equivalent composed of an epidermis containing both melanocytes and keratinocytes. Its dermal compartment consisted either of adipose tissue-derived stromal cells (ASC), dermal fibroblasts, or a mixture of both cell types. These skin substitutes were transplanted for five weeks on the backs of immuno-incompetent rats and analyzed. Gene expression and western blot analyses showed a significantly higher expression of transforming growth factor-β1 (TGF-β1) by ASC in comparison to dermal fibroblasts. In addition we showed that melanocytes responded to the increased levels of TGF-β1 by down-regulating the expression of key melanogenic enzymes such as tyrosinase. This caused decreased melanin synthesis and consequently greatly reduced pigmentation of melDESS. The conclusions are of utmost clinical relevance, namely that that ASC derived from the hypodermis fail to appropriately interact with epidermal melanocytes thus preventing the sustainable restoration of the patient's native skin color in bio-engineered skin grafts.