Abstract: Objective To study the surface markers, differentiation pathways and other properties of mice CD8⁺NK1.1⁺NKT cells amplified in vitro. Methods In vitro amplified mice CD8⁺NK1.1⁺NKT cells were obtained by treating mice spleen cells with Staphylococcus enterotoxin B （SEB）. After the cells were stained with fluorescent antibodies, their surface markers and differential pathways were detected with a flow cytometer（Calibue BD, USA）. Gene transcription levels in cytokines and Foxp3 of the cells were measured by RT-PCR. Results Of the in vitro-amplified cells, CD8⁺T cells accounted for 91.92% with 22.75% being CD8⁺NK1.1⁺ NKT cells, which was 108 times higher than that in normal lymphocytes, and NKT cells accounted for 19.61% of TcRVβ8⁺ NN1.1⁺NKT cells. The expression rate of CD69 was increased from 0.11% to 85.95%. The expression of CD4⁺T, CD8⁺T, CD3⁺NKT, CD4⁺NKT and CD4-CD8-NKT cell subsets did not increase. The mRNA was positively expressed in amplified TGF-β cells which did not express Foxp3 and cytokines including IL-2, 4-6, 10 and IFN-γ. CD8⁺NKT cells were directly differentiated from CD8⁺T cells. Conclusion CD8⁺NK1.1⁺NKT cells, characterized by CD69⁺Foxp3-TcRVβ8⁺ TGF-β⁺CD8⁺NK1.1⁺, are neither CD8⁺ regulatory cells（Treg,） nor CD4⁺NKT cells. They are directly differentiated from CD8⁺T cells and carry TCRVβ8 receptors, thus belonging to a subset of T lymphocytes.