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Chemokines and their receptors play major roles in numerous physiological and pathological processes during development and disease. CXCR4 is the most abundantly expressed chemokine receptor during development. In contrast to other chemokine receptors, CXCR4 binds and is activated exclusively by its ligand stromal derived factor‐1 (SDF‐1) or CXCL12. SDF‐1 signaling has a wide range of effects on CXCR4‐expressing cells depending on the cell type ranging from cell growth to adhesion, chemotaxis, and migration. CXCR4 also serves as a co‐receptor for HIV‐1 entry into T‐cells and has been implicated in the pathogenesis of rheumatoid arthritis and cancer growth and invasion. Numerous inhibitors and antagonists of CXCR4 have been produced and are being tested for their efficiency to target its role in pathogenesis. Our initial expression analysis revealed that CXCR4 is expressed by the migrating myogenic and angiogenic precursors in the developing chick limb. In this study, we used the most specific peptidic inhibitors of CXCR4, T140 and its analog TN14003, to analyse the effect of blocking CXCR4/SDF‐1 signaling on the undetermined bioptent migratory progenitors in the developing chick limb. Our results point to defects in migration and an altered differentiation program of these CXCR4‐expressing progenitor pool in the limb. Developmental Dynamics 235:3007–3015, 2006. © 2006 Wiley‐Liss, Inc.