The efficacy of various combinations of stem cell factor (SCF), FLT3 ligand, interleukin (IL)-2, IL-7 and IL-15 to induce and expand cord blood-derived cytokine-induced killer (CIK) cells was investigated. There were three treatment groups: group A: SCF combined with IL-2, IL-7 and IL-15; group B: SCF, FLT3 ligand combined with IL-2, IL-7 and IL-15, and group C: IL-2, IL-7 and IL-15, the control group. Proliferation rates of CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) natural killer (NK) cells were highest in group B; expansion of CIK cells increased 796.1 ± 278.5-fold, and that of NK cells increased 36.6 ± 3.5-fold. All expanded cord blood-derived CIK/NK cells showed cytotoxic activity against the K562 cell line. Interestingly, the cytotoxicity of group A was highest and significantly higher than that of other groups. These protocols might provide an alternative choice for CIK/NK cell expansion.