In a previous study, we demonstrated that beta1,3-N-acetylglucosaminyltransferase 5 (B3gnt5) is a lactotriaosylceramide (Lc(3)Cer) synthase that synthesizes a precursor structure for lacto/neolacto-series glycosphingolipids (GSLs) in in vitro experiments. Here, we generated B3gnt5-deficient (B3gnt5(-/-)) mice to investigate the in vivo biological functions of lacto/neolacto-series GSLs. In biochemical analyses, lacto/neolacto-series GSLs were confirmed to be absent and no Lc(3)Cer synthase activity was detected in the tissues of these mice. These results demonstrate that beta3GnT5 is the sole enzyme synthesizing Lc(3)Cer in vivo. Ganglioside GM1, known as a glycosphingolipid-enriched microdomain (GEM) marker, was found to be up-regulated in B3gnt5(-/-) B cells by flow cytometry and fluorescence microscopy. However, no difference in the amount of GM1 was observed by TLC-immunoblotting analysis. The GEM-stained puncta on the surface of B3gnt5(-/-) resting B cells were brighter and larger than those of WT cells. These results suggest that structural alteration of GEM occurs in B3gnt5(-/-) B cells. We next examined whether BCR signaling-related proteins, such as BCR, CD19, and the signaling molecule Lyn, had moved into or out of the GEM fraction. In B3gnt5(-/-) B cells, these molecules were enriched in the GEM fraction or adjacent fraction. Moreover, B3gnt5(-/-) B cells were more sensitive to the induction of intracellular phosphorylation signals on BCR stimulation and proliferated more vigorously than WT B cells. Together, these results suggest that lacto/neolacto-series GSLs play an important role in clustering of GEMs and tether-specific proteins, such as BCR, CD19, and related signaling molecules to the GEMs.