Our previous studies demonstrated a causal relationship between GD2/GD3 gangliosides and maintenance of breast cancer stem cells (BCSCs) properties, based on reversal of stem cells properties following knockdown of GD2/GD3 synthases. Recently, we analyzed growth factor receptors (GFRs) associated with GD2/GD3 and their related signaling pathways to clarify how interactions with GD2/GD3 maintain stem cell properties and signaling in BCSCs. Immunofluorescence staining indicated that most GD3 is co-localized with epidermal growth factor receptor (EGFR). In contrast, GD2 and cMet showed only partial colocalization in the ER-Golgi intermediate compartment. Interactions between GD2/GD3 and GFRs were also detected by co-immunoprecipitation of BCSC lysates with anti-GD2 and anti-GD3 antibodies. We observed that there was strong interaction between GD3 and EGFR but weak GD2/ cMet association. On the other hand, β1-integrin, which modulates functions of various GFRs, was strongly associated with GD2. Immunogold labeling and transmission electron microscopy confirmed the localization of GD2/GD3 and GFRs in membrane microdomains. GD3 was clustered with EGFR in electron-dense membrane patches, whereas GD2 and cMet showed little colocalization on BCSC membrane sheets. EGFR expression and ERK signaling were increased in BCSC populations showing high GD2/GD3 expression. Co-immunoprecipitation with a chemical cross-linker in living cells was used to identify protein complexes associated with GD2/GD3. A nano-LC-MS/MS showed there was a total of 143 GD2-associated and 320 GD3-associated proteins. Our findings provide an important basis for further studies of functional molecular interactions that maintain stem cell properties in BCSCs.