Abstract: Objective Pulmonary stenosis （PS） is one of the most common subtypes of congenital heart disease. Using bioengineered tissue materials to expand the pulmonary artery is one of the current treatments for PS. Cell-coating biomaterials have the best therapeutic effect, but it's a critical problem that the source of pulmonary artery smooth muscle cells for bioengineering tissue is rare. In this study, chemical factor induction methods were used to explore the ability of human embryonic stem cells （hESC） differentiating into pulmonary artery smooth muscle cells. Methods hESC line H9 was culture and changed to lateral mesoderm （LM） and neural ectoderm （NE） inducing medium respectively when the cell confluent reached to 10%. Real-time PCR, Western blot, and cellular immunofluorescence were used to detect the ability of H9 to differentiate into vascular smooth muscle cells （VSMC）. Results H9 cells grew in a clone-like manner, SOX2, OCT4, and NANOG were all positively expressed. The expressions of ISL-1, NKX2.5, KDR （marker genes of LM）, Nesting, PAX6, GBX2 （marker genes of NE cells） were up-regulated while the hESC marker gene SOX-2, OCT-4 expressions were down-regulated after induction compared with control group. These two type of cells can further differentiate into VSMCs with α-SMA, CNN1, MYH11, TAGLN, and SMTN-A positive. Conclusion hESC can differentiate into pulmonary artery smooth muscle like cells under specific inducing condition.