Epithelial-mesenchymal interactions promote the morphogenesis and homeostasis of human skin. However, the role of the basement membrane (BM) during this process is not well-understood. To directly study how BM proteins influence epidermal differentiation, survival and growth, we developed novel 3D human skin equivalents (HSEs). These tissues were generated by growing keratinocytes at an air-liquid interface on polycarbonate membranes coated with individual matrix proteins (Type I Collagen, Type IV Collagen or fibronectin) that were placed on contracted Type I Collagen gels populated with dermal fibroblasts. We found that only keratinocytes grown on membranes coated with the BM protein Type IV Collagen showed optimal tissue architecture that was similar to control tissues grown on de-epidermalized dermis (AlloDerm) that contained intact BM. In contrast, tissues grown on proteins not found in BM, such as fibronectin and Type I Collagen, demonstrated aberrant tissue architecture that was linked to a significant elevation in apoptosis and lower levels of proliferation of basal keratinocytes. While all tissues demonstrated a normalized, linear pattern of deposition of laminin 5, tissues grown on Type IV Collagen showed elevated expression of α6 integrin, Type IV Collagen and Type VII Collagen, suggesting induction of BM organization. Keratinocyte differentiation (Keratin 1 and filaggrin) was not dependent on the presence of BM proteins. Thus, Type IV Collagen acts as a critical microenvironmental factor in the BM that is needed to sustain keratinocyte growth and survival and to optimize epithelial architecture.