Objective: During recent decades hematopoietic stem cell transplantation has achieved a significant progress in treatment of many hematologic malignancies. Cord blood as a rich source of hematopoietic stem cells has many advantages than other sources. Because of low number of HSCs in the samples, there is an important limitation in using cord blood stem cells in adults. Expansion of cord blood HSCs in vitro is a solution to overcome this limitation. Mesenchymal stem cells (MSCs) as stromal cell would support proliferation of cord blood HSCs in culture media. It seems that a three-dimensional culture media coated by these mesenchymal stam cells can increase proliferation of cell by creating a microenvironment like that of in-vivo. Materials and Methods: Mononuclear cells from bone marrow were isolated by density gradient centrifugation. Cells were cultured overnight and the adherent cells were allowed to attach to the flask. Non adherent cells were removed and other adherent cells were kept for 10-14 days. Umbilical cord bloods (UBC) CD34+ HSCs were isolated with immunomagnetic separation system. UCB CD34+ cells were expanded for 14 days in three conditions: 1. stroma free culture medium containing three cytokines (TPO, SCF, FL); 2. two dimensiona condition contains MSCs co-culture with cytokines; 3. three dimensional condition contains MSCs co-culture with cytokines on calcium phosphate scaffold. On days 3, 7 and 14 aliquots of cultured cells were harvested and subjected to cell count, colony forming cell assay and flowcytometric analysis. Results: In the stroma free culture media the mean of fold increase of CD34+, number of colony forming unit and the percentage of CD34+ cells after 14 days were 31 ± 2.4, 31 ± 4 and 10.56 ± 4.3% respectively. However this information for 2D co-culture with MSCs after 14 days was 389 ± 18, 305 ± 4 and 67.28 ± 7.4%, and for 3D co-culture with MSCs after 14 days were 247 ± 13, 186 ± 5 and 41.46 ± 7.1%. Conclusion: The results showed that 2D and 3D co-cultures led to significantly higher expansion of UCB HSCs than cultures without MSCs and supplemented only with cytokines.