The mesenchymal tissues involved in cardiac septation are derived from different sources. In addition to endocardial-derived mesenchyme, the heart also receives contributions from the neural crest, the proepicardium, and the dorsal mesenchymal protrusion (DMP). Whereas the contributions of the neural crest and proepicardium have been thoroughly studied, the DMP has received little attention. Here, we present the results of a comprehensive spatiotemporal study of the DMP in cardiac development. Using the Tie2-Cre mouse, immunohistochemistry, and AMIRA reconstructions, we show that the DMP, in combination with the mesenchymal cap on the primary atrial septum, fuse with the major atrioventricular cushions to close the primary atrial foramen and to form the atrioventricular mesenchymal complex. In this complex, the DMP constitutes a discrete prominent mesenchymal component, wedged in between the major cushions. This new model for atrioventricular septation may provide novel insights into understanding the etiology of congenital cardiac malformations. Developmental Dynamics 236:1287–1294, 2007. © 2007 Wiley-Liss, Inc.