AbstractGalectin-3 (Gal-3) is a β-galactoside binding protein that controls cell-cell and cell-extracellular matrix interactions. In lymphoid organs, gal-3 inhibits B cell differentiation by mechanisms poorly understood. The B cell development is dependent on tissue organization and stromal cell signaling, including IL-7 and Notch pathways. Here, we investigate possible mechanisms that gal-3 interferes during B lymphocyte differentiation in the bone marrow (BM) and spleen. The BM of gal-3-deficient mice (Lgals3^−/− mice) was evidenced by elevated numbers of B220⁺CD19⁺c-Kit⁺IL-7R⁺ progenitor B cells. In parallel, CD45⁻ bone marrow stromal cells expressed high levels of mRNA IL-7, Notch ligands (Jagged-1 and Delta-like 4), and transcription factors (Hes-1, Hey-1, Hey-2 and Hey-L). The spleen of Lgals3^−/− mice was hallmarked by marginal zone disorganization, high number of IgM⁺IgD⁺ B cells and CD138⁺ plasma cells, overexpression of Notch ligands (Jagged-1, Delta-like 1 and Delta-like 4) by stromal cells and Hey-1. Morever, IgM⁺IgD⁺ B cells and B220⁺CD138⁺ CXCR4⁺ plasmablasts were significantly increased in the BM and blood of Lgals3^−/− mice. For the first time, we demonstrated that gal-3 inhibits Notch signaling activation in lymphoid organs regulating earlier and terminal events of B cell differentiation.