Objective: Mesenchymal stem cells (MSC) are promising candidates for cell-based therapies. One major obstacle for their clinical use is the biosafety of fetal calf serum (FCS), which is a crucial part of currently used media for the culture of MSC. Materials and Methods: UC-MSC was isolated by cutting the umbilical cord into 1 mm3 fragments and then put the fragments in α-MEM medium with 10% FBS, filtered FFP (10%, 5%, 3% and 1%), filtered and inactivated FFP (10%, 5%, 3% and 1%) respectively. UCMSCs express the pluripotency markers Oct-4, Sox-2, and Nanog by PCR. The cells were CD90, CD105 and CD44 positive by flow Cytometry and could be induced to mesodermal lineages including osteogenic and adipogenic differentiation. Results: Isolation of MSC after 3 days appeared best in media containing 10% FBS and after that with 1% filtered FFP. The medium of other concentration of FFP showed gelatinous change and the cells finally detached from the plate. Conclusion: One percent filtered FFP appears at least as good as 10% FCS with regard to both isolation and expansion of human MSC, while 5%,3% and 10% appear inferior.