The co-receptor CD28 binds to several intracellular proteins including PI3 kinase, Grb-2, Gads and ITK. Grb-2 and PI3 kinase binding has been mapped to the pYMNM motif within the cytoplasmic tail of CD28 and has been shown to play a role in co-stimulation. In this study, we demonstrate that amongst the Grb-2 family adapter proteins, CD28 precipitated Grb-2 and specifically co-operated in the up-regulation of NEAT/AP-1 transcription. By contrast, Gads and Grap either failed or only weakly collaborated with CD28 ligation. Further, the loss of Grb-2 binding interferes with the ability of Vav1 to co-operate with CD28. Anti-CD28 ligation alone was capable for co-operating with Grb-2 or Grb-2-Vav1. Our findings define a pathway involving CD28 binding to Grb-2 and its co-operativity with Vav1 in the regulation of T-cell co-stimulation. 2008 Elsevier Inc. All rights reserved.