Creation of bioartificial organs has been enhanced by the development of strategies involving decellularized mammalian lung. Because fibroblasts critically support lung function through a number of mechanisms, study of these cells in the context of the decellularized lung has the potential to improve the structure and function of tissue-engineered lungs. We characterized the engraftment and survival of a mouse fibroblast cell line in decellularized rat lung slices and found a time-dependent increase in cell numbers assessed by hematoxylin and eosin staining, cell proliferation assessed by Ki67 staining, and minimal cell death assessed by TUNEL staining. We developed a repopulation index to allow quantification of cell survival that accounts for variation in cell density throughout the seeded scaffold. We then applied this method to the study of mouse lung scaffolds and found that decellularization of presliced mouse lungs produced matrices with preserved alveolar architecture and proteinaceous components including fibronectin, collagens I and IV, laminin, and elastin. Treatment with a β1-integrin-neutralizing antibody significantly reduced the repopulation index after 24 h of culture. Treatment with focal adhesion kinase (FAK) inhibitor and extracellular signal-regulated kinase (ERK) inhibitor further reduced initial repopulation scores while treatment with AKT inhibitor increased initial scores. Rho-associated kinase inhibitor had no discernible effect. These data indicate that initial adhesion and survival of mouse fibroblasts in the decellularized mouse lung occur in a β1-integrin-dependent, FAK/ERK-dependent manner that is opposed by AKT.