Developing synthetic lung surfactants to replace animal extracts requires a fundamental understanding of the roles of the various lipids and proteins in native lung surfactant. We used Brewster angle microscopy (BAM), atomic force microscopy (AFM), and Langmuir isotherms to study the influence of palmitoyloleoylphosphatidylglycerol (POPG) in monolayers of dipalmitoylphosphatidylcholine and palmitic acid mixtures with or without dSP-B1-25 a peptide dimer based on the first 25 amino acids of surfactant protein B (SP-B). At surface pressures between 30 and 40 mN/m, only monolayers containing POPG and dSP-B1-25 showed plateaus in the isotherm similar to those in Survanta, a bovine extract replacement lung surfactant that contains native SP-B and SP-C proteins. BAM images show distinct morphological changes in the fluid phase during these plateaus, while AFM images of deposited monolayers show that multilayer structures, which we named "nanosilos", form in the fluid phase at the plateau. These nanosilos are from 50 to 300 nm in diameter and from 5 to 8 nm in height and are similar to those observed in deposited Survanta monolayers. We propose that POPG and SP-B interact to stabilize the monolayer composition by trapping POPG in three-dimensional surface-associated aggregates at high surface pressures, preventing the irreversible loss of POPG and SP-B to the subphase.