A current challenge in cell motility studies is to understand the molecular and physical mechanisms that govern chemokine receptor nanoscale organization at the cell membrane， and their influence on cell response. Using single-particle tracking and super-resolution microscopy， we found that the chemokine receptor CXCR4 forms basal nanoclusters in resting T?cells， whose extent， dynamics， and signaling strength are modulated by the orchestrated action of the actin cytoskeleton， the co-receptor CD4， and its ligand CXCL12. We identified three CXCR4 structural residues that are crucial for nanoclustering and?generated an oligomerization-defective mutant that dimerized but did not form nanoclusters in response to CXCL12， which severely impaired signaling. Overall， our data provide new insights to the field of chemokine biology by showing that receptor dimerization in the absence of nanoclustering is unable to fully support CXCL12-mediated responses， including signaling and cell function in?vivo.