Abstract: In order to explore the regulation mechanisms of follicular helper T cell(Tfh Cell)differentiation, optimized conditions of in vitro induction from both peripheral blood mononuclear cells and MAC sorted Naï ve CD4⁺ T cells to human Tfh cells were developed. Induction efficiency difference of TCR signal anti-hCD3e stimulation between coated on solid phase and in soluble phase was also determined. Differentiation efficiency of CD4⁺CXCR5⁺ICOS⁺PD-1⁺ Tfh cell was determined by FACS while the expression level of IL-21 in cell supernatant was determined by ELISA tests. An ultimate induction condition that 5 μg/mL coated overnight anti-hCD3e stimulated naï ve CD4⁺ T cells to differentiate into Tfh at an up to 20. 4% percentage was finally determined. The optimization of in vitro induction protocol of human Tfh provided an effective examine platform for the studies on Tfh differentiation mechanisms and related pharmacology, toxicity and metabolic experiments.