GM3 and GD3 are the 2 main gangliosides present in the outer layer of bovine milk fat globule membranes (MFGM). Lipids in the outer layer of the MFGM can condense into liquid-ordered phases known as microdomains, but the role GM3 and GD3 have on the formation of MFGM microdomains is unknown. Furthermore, studies have not described the microdomains formed in lipid layers where both GM3 and GD3 are present, as they are in the MFGM. In this study, Langmuir-Blodgett films were made from lipid monolayers containing cholesterol, buttermilk phosphatidylcholine, buttermilk sphingomyelin, and milk gangliosides GM3 and GD3. Lipids were spread at the air-water interface on a 740 mm x 75 mm high-compression Langmuir trough, and Langmuir-Blodgett films were analyzed using a 3-dimensional laser scanning confocal microscope. Observed microdomains were analyzed for shape and size among lipid monolayers of varying ratios of the lipids studied. Understanding how the major gangliosides in milk affect microdomains in the MFGM allows us to understand the structural role of GM3 and GD3 in milk.