Inflammation triggered by microbial lipopolysaccharide (LPS) through Toll‐like receptor (TLR) 4 in the presence of interferon (IFN)‐γ induces cytokine secretion in dendritic cells (DCs) tightly regulated by a defined differentiation program. This DC differentiation is characterized not only by a dynamic immune activating but also by tolerance‐inducing phenotype associated with down‐modulation of cytokines previously considered to be irreversible. CD40L on activated T cells further modifies DC differentiation. Using DNA micro‐arrays, we showed down‐regulated mRNA levels of TLR signalling molecules, whereas CD40/CD40L signalling molecules were up‐regulated at a time when LPS/IFN‐γ‐activated DCs had ceased cytokine expression. Accordingly, we demonstrated that CD40/CD40L but not TLR4 or TLR3 signalling mediated by LPS or poly (cytidylic‐inosinic) acid (poly I:C) and dsRNA re‐established the capacity for secreting interleukin (IL)‐12 in primarily LPS/IFN‐γ‐activated DCs, which have exhausted their potential for cytokine secretion. The resulting TH1 polarizing DC phenotype – which lacked accompanying secretion of the crucial immune suppressive factor IL‐10 – maintained the potential for activation of cytotoxic T lymphocytes (CTLs). We therefore conclude that immune modulation is restricted to a secondary T‐cell‐mediated stimulus at an exhausted DC state, which prevents an immune tolerant DC phenotype. These findings impact on the rational design of TLR‐activated DC‐based cancer vaccines for the induction of anti‐tumoural CTL responses.