Optimal activation of Rel / NF‐κB transcription factors in T lymphocytes requires a CD28‐delivered co‐stimulatory signal in addition to TCR engagement. Although, Rel / NF‐κB transcription factors are critical regulators of many T cell functions, the mechanisms and molecules, which link the surface receptors to their activation, are poorly characterized. Using Jurkat T cells stimulated with superantigen presented on B7‐positive APC, we showed that CD28‐ and TCR‐stimulated NF‐κB‐dependent transcription is associated to the activation of IκB kinase β (IKKβ) and, to a lesser extent, of IκB kinase α (IKKα). A dominant negative mutant of the MAP3 kinase MEKK1, a kinase known to regulate the JNK pathway and to activate NF‐κB‐dependent transcription in many cell types, strongly inhibits CD28‐ and TCR‐induced IKK activity, whereas the dominant negative mutants of the NF‐κB‐inducing kinase (NIK) did not exert any significant effects. In addition, TCR / CD28 stimulation results in the recruitment and autophosphorylation of endogenous MEKK1, whereas endogenous NIK was not detectably activated. Our data identify MEKK1 as a critical step in coupling signals initiated by TCR and CD28 to the downstream pathways which lead to both AP‐1 and NF‐κB activation in T lymphocytes.