Differentiation of T lymphocytes is a complex and finely tuned process. Here we show that treatment of mouse fetal thymus organ cultures with agents activating the cAMP‐dependent signalling pathway results in the block of thymocyte differentiation. This is due to severe impairment of maturation beyond the CD4‐/CD8‐ stage. In addition, rearrangements at the TCR alpha gene locus, but not at the TCR beta locus, are completely inhibited. The cAMP effect is reversible and is restricted to TCR alpha beta+ cells. cAMP acts both by triggering apoptosis and by inducing cell‐cycle block in thymocytes. Thus, activation of the cAMP pathway provides a mechanism to modulate thymic function for hormones and ligands whose receptors are coupled to adenylate cyclase.