A method of differentiating pluripotent stem cells into mesenchymal stem cells, a culture medium used in the method of differentiating pluripotent stem cells into mesenchymal stem cells and a method of performing tissue and organ regeneration by using the mesenchymal stem cells obtained by differentiation using the method of differentiating pluripotent stem cells into mesenchymal stem cells are provided. The method of differentiating pluripotent stem cells into mesenchymal stem cells comprises differentiating, completely under 3D suspension conditions, pluripotent stem cells into trophoblast-like cells using BMP4 and A8301, and then differentiating the trophoblast-like cells into mesenchymal stem cells. Neither of two differentiation processes needs passaging or replacement of a culture container.