Human cord blood (CB), collected from the postpartum placenta and umbilical cord, has been identified as a rich source of hematopoietic stem cells (HSCs) and provides an alternative to bone marrow or mobilized peripheral blood transplantation. However, the major restriction of CB transplantation is the low number of HSCs in each CB unit and limits its use in clinical transplantation. The development of ex vivo culture systems that facilitate the expansion of HSCs is crucial to stem cell research and clinical application. In this chapter, we describe the protocols to isolate HSCs from CB, expand HSCs in serum-free condition, and analyze HSCs. This information is benefical for successful use of CB stem cells in therapeutic studies.