Background: Mesenchymal stem cells (MSCs) are multipotent progenitors capable of differentiation to not only mesodermal tissues including bone, cartilage, and muscle but also adipoid, hematopoietic, and neural cells, which makes MSC an attractive candidate for clinical applications. We have previously reported optimal culture conditions for the stepwise differentiation of human embryonic stem cells to unlimited numbers of pure Multipotent Mesenchymal Precursors (MMPs) from human Embryoid bodies (hEBs). Here, we demonstrate more standardized and simple method to reproducibly differentiate hESCs/EB-derived MMPs. Methods and Results: We derivates MMPs from two human Embryonic Stem Cell lines(SNUhES3 & CHA-3) with the standardized size of EBs and various cytokine-based mesodermal lineage commitment condition. EBs formed in serum-free medium without bFGF for 14 days ware attached in gelatin coated culture dish with DMEM supplemented with 10% FBS and they were trypsinized at day 16 and were incubated with BMP based conditioned medium. The differentiated cell lines obtained with this procedure are morphologically similar to bone marrow MSCs and can grow stably in vitro culture for about 40 passages. They showed immunophenotype similar to bone marrow MSCs; negative for CD34 and CD45 and positive for CD44 (HCAM), SH2 (CD105), SH3 (CD73). Furthermore they can differentiate into multiple cell lineages including osteocytes, chondrocytes, and adipocytes and can be used as feeder cells to support the growth of undifferentiated hESCs. Finally, we confirmed that hESCs/EBs-derived MMPs did not form tumor until 4 months' observation after transplantation to the immunotolerant SCID mice. Conclusion: This simple and standardized protocol will provide more effective platform to produce large amounts of genetically identical MSCs that are promising candidates in clinical application.