Follicular helper T (Tfh) cells are crucial for humoral immunity. Dysregulation of Tfh cell differentiation can cause infectious, allergic, and autoimmune diseases. To elucidate the molecular mechanisms underlying Tfh cell differentiation, we attempted to establish an in vitro mouse model of Tfh cell differentiation in the absence of other cell types. Various cytokines and cell surface molecules are suggested to contribute to the differentiation. We found that stimulating naïve CD4+T cells with immobilized antibodies to CD3, ICOS, and LFA-1 in the presence of soluble anti-CD28 antibody, IL-6, and antibodies that block IL-2 signaling for 3 days induced the expression ofBcl6andRorc(γt), master regulator genes of Tfh and Th17 cells, respectively. TGF-β significantly enhanced cell proliferation andBcl6andRorc(γt)expression. An additional 2 days of culture without immobilized antibodies selectively downregulatedRorc(γt)expression. These cells produced IL-21 and promoted B cells to produce IgG antibodies. Adding the aryl hydrocarbon receptor (AhR) antagonist CH-223191 to the T cell culture further downregulatedRorc(γt)expression without significantly affectingBcl6expression, and upregulated expression of a key Tfh marker, CXCR5. Although their CXCR5 expression levels were still not high, the CH-223191-treated cells showed chemotactic activity towards the CXCR5 ligand CXCL13. On the other hand, AhR agonists upregulatedRorc(γt)expression and downregulated CXCR5 expression. These findings suggest that AhR activity and the duration of T cell receptor stimulation contribute to regulating the balance between Tfh and Th17 cell differentiation. Although this in vitro system needs to be further improved, it may be useful for elucidating the mechanisms of Tfh cell differentiation as well as for screening physiological or pharmacological factors that affect Tfh cell differentiation including CXCR5 expression.