Organ morphogenesis, including lung morphogenesis, involves a series of cellular behaviors that are difficult to observe and document in vivo due to current limitations in imaging techniques. Therefore, in vitro models are necessary to study these cellular behaviors as well as basic developmental processes relevant to in vivo morphogenesis. Here, we describe a novel in vitro three-dimensional (3D) culture system for assessing mouse lung alveolar morphogenesis using primary fetal mouse lung cells cultured in Matrigel supplemented with fibroblast growth factor 10 and hepatocyte growth factor. In our in vitro 3D culture system, single primary mouse fetal lung cells successfully grew, developed lumen, and formed multivesicular epithelial structures, resulting in a morphology that was highly similar to that of lung alveoli. This culture system is a useful tool for investigating the cellular and molecular mechanisms involved in lung alveolar morphogenesis.