Tissue engineering is a promising approach to treat massive airway dysfunctions such as tracheomalacia or tumors. Currently, there is no adequate solution for patients requiring the resection of more than half of the length of their trachea. In this study, the best conditions for combination of three different cell types from the respiratory airway system were investigated to develop a functional ciliated and pre‐vascularized mucosal substitute in vitro. Primary human fibroblasts were combined with respiratory epithelial cells and endothelial cells. As scaffolds, fibrin gel and agarose‐type I collagen blends were used and cultured with different medium compositions to optimize both vascularization and differentiation of the respiratory epithelium. A mixture of endothelial growth medium and epithelial differentiation medium was shown to optimize both vascularization and epithelial growth and differentiation. After 28 days of co‐culture, significantly increased formation of capillary‐like structures was observed in fibrin gels with more than three times higher structure volumes compared to agarose–collagen gels. After 35 days, epithelial differentiation into a pseudostratified epithelium with typical marker expression was improved on fibrin gels. While cilia formation was shown on both scaffolds, a higher number of ciliated cells and longer cilia were observed on fibrin gels. The data elucidate the important interplay of co‐culture parameters and their impact on vascularization as well as epithelium development and provide a basis for development of functional three‐dimensional airway constructs.