Hypoxia-inducible factor-1α (HIF-1α) is a key regulator of the physiological response to hypoxia. To study the regulation mechanism of HIF-1α in proliferation and differentiation of stem cells, two small interference RNA expression vectors of HIF-1α were constructed, and transfected into the fetal liver stromal cell lines (FLSCs) stably by lentiviral system. The efficiency of virus transfection was identified by expression of green fluorescence protein(GFP) analyzed by fluorescence microscope, then the high GFP expression FLSCs were sorted by fluorescence-activated cell sorting (FACS) according to strong GFP expression. Analysis of efficiency of RNA interfering on HIF-1α was detected by real time-PCR and Western-blot. The HIF-1α gene expression at mRNA level of FLSCs transfected by lentivirus plasmids pSicoR-HIF-1α-1 and pSicoR-HIF-1α-2 are 18.8% and 25.5% of the FLSCs transfected by the control lentivirus under 20% O₂; 21.2% and 29.3% under hypoxia. The HIF-1α protein were down regulated by siRNA. The pSicoR-HIF1 showed higher interfering efficiency than pSicoR-HIF2. The expression of SDF-1α gene in HIF-1α silencing FLSCs were detected by RT-PCR, fluorescent immunocytochemistry analysis and ELISA. The mRNA and protein of SDF-1α gene were down regulated after silencing of HIF-1α. Therefore, the regulation of SDF-1α gene under hypoxia could be activated by HIF-1α, that plays an important role in the influence on the proliferation and differentiation of stem cell.