Many studies demonstrate that the type of adjacent mesenchymal cells can affect epidermal morphogenesis of bilayered tissue-engineered skin. However, whether a mixture of different mesenchymal cell types can improve epidermal morphogenesis of bioengineered skin remains unknown. In this study, keratinocytes, dermal fibroblasts and adipose tissue-derived stem cells (ADSCs) were isolated and purified from human skin and subcutaneous fat. Conditioned medium generated from a mixture of dermal fibroblasts and ADSCs at the ratio of 1:1 was superior to that from fibroblasts or ADSCs alone in promoting keratinocyte proliferation, as indicated by MTT assay. Furthermore, ELISA results showed that the cytokine levels of human hepatocyte growth factor and keratinocyte growth factor (also known as FGF7) in the mixed fibroblasts/ADSC group were higher than those in the ADSC or dermal fibroblasts group. To examine the potential roles of mixed fibroblasts and ADSCs on epidermal morphogenesis, a three-dimensional tissue engineered skin system was applied. Histological analyses demonstrated that keratinocytes proliferated extensively over the mixture of fibroblasts and ADSCs, and formed a thick epidermal layer with well-differentiated structures. Keratin 10 (epidermal differentiation marker) was expressed in the suprabasal layer of bilayered tissue-engineered skin in the mixed fibroblasts and ADSCs group. Desmosomes and hemidesmosomes were detected in the newly formed epidermis by transmission electron microscopy analysis. Together, these findings revealed for the first time that a mixture of fibroblasts and ADSCs in bilayered tissue-engineered skin can improve epidermal morphogenesis.