Germinal center (GC) dark and light zones segregate cells undergoing somatic hypermutation and antigen-driven selection, respectively, yet the factors guiding this organization are unknown. We report here that GC organization was absent from mice deficient in the chemokine receptor CXCR4. Centroblasts had high expression of CXCR4 and GC B cells migrated toward the CXCR4 ligand SDF-1 (CXCL12), which was more abundant in the dark zone than in the light zone. CXCR4-deficient cells were excluded from the dark zone in the context of a wild-type GC. These findings establish that GC organization depends on sorting of centroblasts by CXCR4 into the dark zone. In contrast, CXCR5 helped direct cells to the light zone and deficiency in CXCL13 was associated with aberrant light zone localization.