Invariant Natural Killer T (iNKT) cells are unique subset of innate T cells that regulate several immune responses, thus have potential for novel immunotherapeutics to modulate adaptive immunity in certain diseases such allergy, infectious, autoimmune disease and cancer. One of the limitations for developing iNKT cell therapy is difficulty to acquire clinical meaningful number of iNKT cells as they are extremely rare population comprising only less than 0.1% of circulating T cells. Here, we developed an effective strategy to expand iNKT cells in Ex Vivo for adoptive cell therapy.  First, iNKT cells from adult peripheral blood mononuclear cells (PBMC) and cord blood mononuclear cells (CBMC) were enriched by MACS with anti-iNKT microbeads, and subsequently co-cultured with allogeneic dendritic cells in the presence of αGalCer and IL-2. After single antigen specific stimulation for 10-14 days, we obtained 2.8x10e7 iNKT cells (range: 0.1-7.0x10e7) from 12 consecutive 5x10e8 adult PMBC, and 1.2x10e7 iNKT cells (range: 0.4-4.9x10e7) from 10 consecutive 5x10e8 CBMC. Although the lower absolute number of iNKT cells was present in cord blood, cord iNKT cells underwent a drastic expansion by 493.7 folds (range:200-1684) compared to 71.2 folds (range:22-3937) for adult iNKT cells. The purity of expanded iNKT cells were consistently greater than 90%. Expanded cord iNKT cells were exclusively CD4+ (97%, range:96.5-99.79%), while adult iNKT cells contained varying degree of CD4+(68.8%, range:38-89%), CD4-CD8alpha-(22.8%, range:4.0-47.9%), and CD8alpha+ (6.2%, range:0.73-17.80%). Expanded cord CD4+iNKT cells showed a profound polarization towards Th2- functional phenotype compared to expanded adult CD4+iNKT cells, and expanded adult CD4-iNKT cells displayed polarizationtowards Th-1 functional phenotype and expressed higher degrees of various natural killer receptors. In summary, we demonstrated that it is feasible to acquire highly pure, and highly polarized iNKT cells through single antigenic expansion in clinically meaningful number for adoptive cell therapy to modulate adaptive immunity in immune-microenvironment.