The biological outcome of receptor-mediated signalling often depends on the duration of engagement. Because CD40 signalling is controlled by the regulated expression of its ligand, CD154, the mechanisms that regulate CD154 expression probably determine the strength and duration of CD40 signalling. Here, we demonstrate that CD154 expression on the surface of mouse CD4 T cells can be separated into an early phase, occurring between 0 and 24 hr after T-cell activation, and a later extended phase, occurring after 24 hr. The early phase of CD154 expression did not require costimulation and was probably influenced by the strength of T-cell receptor (TCR) signalling alone. However, later CD154 expression was highly dependent on costimulation through either CD28 or inducible costimulator (ICOS). Although CD28 signalling interleukin (IL)-2 secretion, ICOS not, suggesting that costimulation enhance CD154 expression independently of IL-2 production. In fact, anti-CD28 treatment could still induce late-phase CD154 on anti-CD3-stimulated CD4 T cells expressing a mutated form of CD28 that not lead to the induction of IL-2. However, this CD154 induction was somewhat weaker than that of wild-type CD28-expressing cells, suggesting that direct signalling and IL-2-mediated signalling co-operatively responsible for the levels of CD154 induced by CD28. Finally, we show that the second phase of CD154 expression negatively regulated B-cell terminal differentiation and antibody secretion. These results demonstrate that TCR signalling and costimulation each regulate different phases of CD154 expression and control the biological outcome of CD40 signalling on B cells.