Interactions between B cell progenitors and bone marrow stromal cells are essential for normal B cell differentiation. We have previously shown that an immune developmental synapse is formed between human pre-B and stromal cells in vitro, leading to the initiation of signal transduction from the pre-BCR. This process relies on the direct interaction between the pre-BCR and the stromal cell-derived galectin-1 (GAL1) and is dependent on GAL1 anchoring to cell surface glycosylated counterreceptors, present on stromal and pre-B cells. In this study, we identify alpha(4)beta(1) (VLA-4), alpha(5)beta(1) (VLA-5), and alpha(4)beta(7) integrins as major GAL1-glycosylated counterreceptors involved in synapse formation. Pre-B cell integrins and their stromal cell ligands (ADAM15/fibronectin), together with the pre-BCR and GAL1, form a homogeneous lattice at the contact area between pre-B and stromal cells. Moreover, integrin and pre-BCR relocalizations into the synapse are synchronized and require actin polymerization. Finally, cross-linking of pre-B cell integrins in the presence of GAL1 is sufficient for driving pre-BCR recruitment into the synapse, leading to the initiation of pre-BCR signaling. These results suggest that during pre-B/stromal cell synapse formation, relocalization of pre-B cell integrins mediated by their stromal cell ligands drives pre-BCR clustering and activation, in a GAL1-dependent manner.