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The functions of a chemokine CXC chemokine ligand (CXCL) 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its physiologic receptor CXCR4 in T cell development are controversial. In this study, we have genetically further characterized their roles in fetal and adult T cell development using mutant and chimeric mice. In CXCL12−/− or CXCR4−/− embryos on a C57BL/6 background, accumulation of T cell progenitors in the outer mesenchymal layer of the thymus anlage during initial colonization of the fetal thymus was comparable with that seen in wild-type embryos. However, the expansion of CD3− CD4− CD8− triple-negative T cell precursors at the CD44− CD25+ and CD44− CD25− stages, and CD4+ CD8+ double-positive thymocytes was affected during embryogenesis in these mutants. In radiation chimeras competitively repopulated with CXCR4−/− fetal liver cells, the reduction in donor …