The simultaneous expansion and harvest of hematopoietic stem cells and mesenchymal stem cells derived from umbilical cord blood were carried out using bioreactors. The co-culture of umbilical cord blood (UCB)-derived hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) was performed within spinner flasks and a rotating wall vessel (RWV) bioreactor using glass-coated styrene copolymer (GCSC) microcarriers. The medium used was composed of serum-free IMDM containing a cocktail of SCF 15 ng·mL(-1), FL 5 ng·mL(-1), TPO 6 ng·mL(-1), IL-3 15 ng·mL(-1), G-CSF 1 ng·mL(-1) and GM-CSF 5 ng·mL(-1). Accessory stromal cells derived from normal allogeneic adipose tissue were encapsulated in alginate-chitosan (AC) beads and used as feeding cells. The quality of the harvested UCB-HSCs and MSCs was assessed by immunophenotype analysis, methylcellulose colony and multi-lineage differentiation assays. After 12 days of culture, the fold-expansion of total cell numbers, colony-forming units (CFU-C), CD34(+)/CD45(+)/CD105(-) (HSCs) cells and CD34(-)/CD45(-)/CD105(+) (MSCs) cells using the RWV bioreactor were (3.7 ± 0.3)- , (5.1 ± 1.2)- , (5.2 ± 0.4)- , and (13.9 ± 1.2)-fold respectively, significantly better than those obtained using spinner flasks. Moreover, UCB-HSCs and UCB-MSCs could be easily separated by gravity sedimentation after the co-culture period as only UCB-MSCs adhered on to the microcarriers. Simultaneously, we found that the fibroblast-like cells growing on the surface of the GCSC microcarriers could be induced and differentiated towards the osteoblastic, chondrocytic and adipocytic lineages. Phenotypically, these cells were very similarly to the MSCs derived from bone marrow positively expressing the MSCs-related markers CD13, CD44, CD73 and CD105, while negatively expressing the HSCs-related markers CD34, CD45 and HLA-DR. It was thus demonstrated that the simultaneous expansion and harvest of UCB-HSCs and UCB-MSCs is possible to be accomplished using a feasible bioreactor culture system such as the RWV bioreactor with the support of GCSC microcarriers.