Factors governing the entry of cells into the postnatal thymus are poorly understood. We aimed to define molecular mechanisms mediating the homing of bone marrow cells to the thymus using a sublethally irradiated in vivo murine model. Entry of unfractionated and lineage-depleted bone marrow cells to the thymus, but not bone marrow, was a Galphai-mediated phenomenon. Lineage-depleted cells that had homed to the thymus expressed abundant CXCR4 and CCR5 mRNA, alone of 17 chemokine receptors evaluated by QPCR. Thymic-homed cells were distinct from cells that had homed to bone marrow in expression of CXCR4 and CCR5 by mRNA quantification and cell-surface expression of protein. Abrogation of CXCR4 and CCR5 function by genetic, antibody, or pharmacologic means impaired homing of lineage-depleted cells to the thymus, although not in a synergistic manner, implying interdependency of these receptors in the homing process. Competitive repopulation experiments demonstrated that inhibiting CXCR4-mediated homing adversely affected the double-negative cell pool at 2 weeks, suggesting that cells with prothymocytic activity may in part home via CXCR4. Overall, our data demonstrate differential homing mechanisms governing entry of unfractionated and lineage-depleted cells to irradiated bone marrow or thymus, with thymic homing of immature cells being pertussis-sensitive and mediated by the chemokine receptors CXCR4 and CCR5.