Objective To explore the morphological relationship and mechanism of pulmonary endoderm with the development of the prepharyngeal mesenchyme from second heart field during the outflow tract septation in mouse embryonic heart. Methods Both the immunohistochemical and immunofluorescence staining methods were used to observe forkhead box A2(Foxa2), islet-1(ISL-1), patched-1(Ptc1), patched-2(Ptc2), α-smooth muscle actin(α-SMA）and myosin heavy chain(MHC) distribution in serial sections of six mouse embryos each day from embryonic day(ED) 9 to embryonic day(ED) 13. Results At ED 9-ED 9.5, locally thickened ISL-1 positive endoderm in the ventral foregut wall predicted the initiation of pulmonary endoderm differentiation. As soon as the lanryngo-tracheal groove from pulmonary endoderm was initiated, ISL-1 positive cells began to appear in the matrix surrounding pulmonary endoderm. With the elongation of the lanryngo-tracheal groove in the direction of aortic sac from ED 10 to ED 11.5, ISL-1 positive prepharyngeal cells formed a cone-shaped structure centered by pulmonary endoderm, and its ventral end protruded into the cavity of aortic sac to form aorto-pulmonary septum. During the development of lanryngo-tracheal groove, a solid endoderm cord could always be observed at the ventral end of lanryngo-tracheal groove, and pulmonary endoderm of lanryngo-tracheal groove and its solid cord, to which strong Ptc1 and Ptc2 expression were mainly confined, was located in the center of the ISL-1 cone-shaped structure. From ED 12 to ED 13，separation of foregut at the level of outflow tract led to the formation of trachea, endoderm cord no longer be found and Ptc1 and Ptc2 positive expression disappeared in the tracheal epithelial. The density of the ISL-1 positive mesenchyme cells reduced sharply in the ventral to trachea, which gradually stopped to be added to outflow tract, and aortic sac was separated eventually.  Conclusion The differentiation and development of pulmonary endoderm are closely associated with the ISL-1 positive prepharyngeal mesenchyme of second heart field aggregation. The activity of sonic hedgehog(SHH) signaling system is in a high level during the pulmonary endoderm development, the developing pulmonary endoderm probably acts as an organizing center via SHH pathway to induce aggregation of ISL-1 positive cells, and the mechanical tension produced by endoderm growth is also to drive those ISL-1 positive cells migration for the normal morphogenesis of outflow tract.