Hematopoietic stem cell (HSC) migration to stromal cell-derived factor (SDF) -1 correlates with CXC chemokine receptor 4 (CXCR4) expression and bone marrow homing. Activation of Sphingosine-1-Phophate (S1P) receptors (S1P1–5) modulate chemokine induced migration of lymphocytes and HSC. S1P1 partial activation and internalisation after binding of FTY720, a S1P analogue, leads to trapping of lymphocytes in lymphoid tissue. To analyze the influence of S1P1 on SDF-1 dependent migration in HSC we cloned S1P1 cDNA into an oncoretroviral vector containing an EMCV-IRES GFP reporter gene (MFGS-S1P1-IRES-EGFP). Transduction of mobilized peripheral blood CD34+ hematopoietic stem cells (PBSC) and Jurkat cells with MFGS-S1P1-IRES-EGFP derived virus particles resulted in transgene overexpression in more than 95% of target cells. A significantly higher amount of S1P1 transgene positive PBSCs acquired an adherent phenotype compared to GFP control-transduced cells. Flow cytometric analyses revealed a 3 fold downregulation of CXCR4 expression on S1P1 overexpressing PBSCs and Jurkat cells. SDF-1 dependent migration of S1P1 overexpressing PBSCs or Jurkat-cells in transwell assays was inhibited by more than 90% compared to control transduced cells. In contrast retrovirus mediated overexpression of S1P2 or S1P3 in Jurkat cells did not show a significant influence on SDF-1/CXCR4 dependent migration. SDF-1 dependent ERK1/2 phosphorylation was abolished in S1P1 overexpressing PBSCs, suggesting that Gi-protein signaling pathways are involved in the inhibition of migration. Sublethally irradiated NOD/SCID mice were transplanted with ex vivo cultured 6 day old PBSC which were either transduced with MFGS-S1P1-IRES-EGFP to 95% or with MFGS-EGFP to 85%. Screening for GFP positive human cells in the mouse bone marrow 18 hours after transplantation revealed a 5 to 10 fold reduction (p<0,01) in homing of MFGS-S1P1-IRES-EGFP transduced cells compared to MFGS-EGFP control transduced cells. Our data suggest that high levels of S1P1 act as a downregulator of CXCR4 expression and as a strong inhibitor of CXCR4 dependent migration in HSCs.