This study was designed to establish a more effective and safe culture system for adoptive immunotherapy by investigating the use of homologous cord blood plasma (HCBP) instead of fetal bovine serum (FBS), which has various limitations including ethical problems for the ex vivo expansion of human umbilical T lymphocytes. Fresh human umbilical mononuclear cell fractions were isolated by Ficoll-Hypaque density centrifugation. Nonadherent mononuclear cell fractions were cultured with anti-CD3 antibody (5 μg/ml), IL-2 (175 U/ml), and either 10% FBS or 10% HCBP. On day 8, the cellular proliferation rate and cell surface markers were assessed. There was no significant difference in proliferation when human umbilical cord blood T lymphocytes were grown in medium supplemented with FBS or HCBP (p > 0.05). In medium containing FBS, the proportion of CD3⁺CD4⁺ (markers for helper T cell), CD3⁺CD8⁺ (cytotoxic T cell), CD3⁺CD25⁺ (activated T cell), CD3⁺CD38⁺ (immature T cell), and CD3⁺CD45RO⁺ (memory T cell) cells was significantly increased (p < 0.05), whereas proportion of CD3⁺CD45RA⁺ (naive T cell) and CD16⁺CD56⁺ (NK cell) cells was significantly decreased (p < 0.05). In HCBP supplemented medium, the proportion of CD3⁺CD8⁺, CD3⁺CD25⁺, CD3⁺CD45RA⁺, and CD3⁺CD45RO⁺ cells was significantly increased (p < 0.05). The proportion of CD3⁺CD4⁺, CD3⁺CD45RO⁺ and CD3⁺CD38⁺ cells was significantly higher, but proportion of CD3⁺CD45RA⁺ and CD3⁺CD8⁺ cells was significantly lower in FBS compared with HCBP supplemented medium (p < 0.05). Our results support the feasibility of ex vivo expansion of human umbilical cord blood T lymphocytes in medium supplemented with HCBP for future adoptive cellular immunotherapy.