CXCL12 and VCAM1 retain hematopoietic stem cells (HSC) in the bone marrow but factors mediating HSC egress from the bone marrow to the blood are not known. Sphingosine-1-Phosphate (S1P) receptor 1 (S1P(1)) is expressed on HSC, and S1P facilitates the egress of committed hematopoietic progenitors from the bone marrow into the blood. We show that the S1P gradient between the bone marrow and the blood, and expression of S1P(1), is essential for optimal HSC mobilization by CXCR4 antagonists including AMD3100, and for the trafficking of HSC during steady state hematopoiesis. We also demonstrate that the S1P(1) agonist, SEW2871, increases AMD3100-induced hematopoietic stem and progenitor cell mobilization. These results suggest the combination of a CXCR4 antagonist and a S1P(1) agonist may prove sufficient for mobilizing HSC in normal donors for transplantation purposes, potentially providing a single mobilization procedure and eliminating the need to expose normal donors to granulocyte colony stimulating factor with its associated side effects.