Tissue engineered skin substitutes are valuable not only as a clinical therapy for chronic wounds and severe traumas, but also as in vitro 3D model systems to investigate wound healing and skin pathogenesis. Current tissue engineered skin substitutes are characterized by a flat interface at the dermal-epidermal junction -a stark contrast to the complex microtopography found in native skin. This topography creates 3D microenvironments that regulate keratinocyte function and cell fate. Recently, we demonstrated that incorporating physical topographic cues into an in vitro bilayered skin substitute creates distinct keratinocyte functional niches. To investigate the roles of these functional microniches, we engineered a multi-dimensional micropatterned dermal-epidermal junction analog that recreates these keratinocyte microenvironments and supports both proliferative and synthetic niches. This novel 3D model system allows for the systematic investigation of important topographic cues as well as cell-cell and cell substrate interactions. Here we use these novel analogs to recreate stem cell patterning in vitro and we evaluate them using whole mount immunofluorescent staining coupled with confocal microscopy. This fully implantable model system will allow us to investigate the role of topography in organogenesis in vivo and design next generation skin substitutes for therapeutic use.