Introduction: Umbilical cord blood (CB) has been identified as a rich source for hematopoietic stem cells (HSCs), and has provided an alternative to bone marrow transplantation. The use of ex vivo expanded cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood (CB) transplantation. The main aim of our study is to find the best culture media and condition to increase number of CD34+/CD38- hematopoietic stem cells in cord blood for transplantation. Material and Methods: Mononuclear cells (MNCs) were seperated from cord blood and cultured in RPMI1640 with 10% fetal calf serum (FCS) or 10% cord blood plasma (CBP) or serum free media (SF). Culture media contained 50ng/ml of Interlukin 6 (IL6), IL3, Thrombopoietin (TPO) Stem cell factor (SCF) and flt3-ligand. Cells were cultured for two weeks and number of CD34+/CD38- cells and total MNCs measured at days 0, 7 and 14. Results: At 14 days culture mean fold of expansion of CD34+ and CD34+/CD38- cells was 20.4 and 57.4 for FCS, 5.6 and 10.3 for SF and 10.8 and 4.7 for CBP culture media. Conclusion: Due to efficacy and predictability of SF media for cell expansion and because of its better safety for allergic reactions and microbial contamination (in comparison to animal products containing media) and enough expansion for clinical applications, we suggest that SF media is better than CBP or SF from clinical points of view.