The impact of cream processing on milk fat globule membrane (MFGM) was assessed in an industrial setting for the first time. Three creams and their derived MFGM fractions from different stages of the pasteurization procedure at a butter dairy were investigated and compared to a native control as well as a commercial MFGM fraction. The extent of cross-linking of serum proteins to MFGM proteins increased progressively with each consecutive pasteurization step. Unresolved high molecular weight aggregates were found to consist of both indigenous MFGM proteins and β-lactoglobulin as well as αs1- and β-casein. With regards to fat globule stability and in terms of resistance towards coalescence and flocculation after cream washing, single-pasteurized cream exhibited reduced sensitivity to cream washing compared to non- and double-pasteurized creams. Inactivation of the agglutination mechanism and the increased presence of non-MFGM proteins may determine this balance between stable and non-stable fat globules.