Abstract CXCL12-induced chemotaxis and adhesion to VCAM-1 decrease as B cells differentiate in the bone marrow. However, the mechanisms that regulate CXCL12/CXCR4-mediated signaling are poorly understood. We report that after CXCL12 stimulation of progenitor B cells, focal adhesion kinase (FAK) and PI3K are inducibly recruited to raft-associated membrane domains. After CXCL12 stimulation, phosphorylated FAK is also localized in membrane domains. The CXCL12/CXCR4-FAK pathway is membrane cholesterol dependent and impaired by metabolic inhibitors of Gi, Src family, and the GTPase-activating protein, regulator of G protein signaling 1 (RGS1). In the bone marrow, RGS1 mRNA expression is low in progenitor B cells and high in mature B cells, implying developmental regulation of CXCL12/CXCR4 signaling by RGS1. CXCL12-induced chemotaxis and adhesion are impaired when FAK recruitment and phosphorylation are inhibited by either membrane cholesterol depletion or overexpression of RGS1 in progenitor B cells. We conclude that the recruitment of signaling molecules to specific membrane domains plays an important role in CXCL12/CXCR4-induced cellular responses.