Cytokine-induced killer (CIK) cells are a heterogeneous population typically including more than 30% of CD3 + CD56 + cells and efficiently kill tumor cells. Here, we provide important technical tips to generate successfully CIK cells from mouse spleen cells. To keep the viability and proliferation of CIK cells, two critical factors were essential: the consecutive addition of IFN-γ, anti-CD3 antibody, and a large enough amount of IL-2; cell density adjustment during the cultivation. On day 14, the cell number increased by > 200-fold, the phenotypes were > 90% CD3 + CD8 + and > 45% CD3 + NK1.1 + . They expressed IFN-γ, IL-2, granzymes, and FasL, and had strong antitumor activity in vitro and in vivo. Taken together, our data provide an optimal protocol for the generation of CIK cells having more than > 30% of CD3 + CD56 + cells.