Basic fibroblast growth factor (bFGF or FGF-2) plays an important role in modulating proliferation, migration, differentiation and survival of various cells, such as fibroblasts, endothelial cells, smooth muscle cells and neural cells. The fetal liver stromal cell lines (FLSCs) expressing bFGF stably has been established by lentiviral system. The FLSCs were isolated from aborted fetus with informed consent, the growth character was identified by MTT method and the surface marker analyzed by flow cytometry. The cells could maintain normal karyotype after passaged for 35 generations. The coding region of 18 ku bFGF was cloned from fetal bone marrow mesenchymal stem cells (BMSCs) by RT-PCR. The bFGF recombinant lentiviral plasmid was steadily transfected into FLSCs. The low and high bFGF-expression FLSCs that transfected by the recombinant lentivirus were sorted by fluorescence-activated cell sorting (FACS) according to weak and strong eGFP expression. Analysis of weak and strong eGFP expression transfected FLSCs by RT-PCR and Western-blot demonstrated the stable expression of bFGF after 15 passages. The bFGF expression at mRNA level of weak and strong eGFP expression FLSCs are 2.33 and 6.19-fold for the FLSCs transfected by the control lentivirus, and then at the protein level are 1.76 and 5.05-fold. hES cells can be matained for 20 passages using the bFGF/FLSCs as feeder layers supplemented with a little or no additional recombinant bFGF. The establishment of bFGF/FLSCs could be acted as feeder cells for sustainment of human embryonic stem cell in vitro by supplying an eligible microenvirnment free of animal feeder layers.