Other family members expressed in thymocytes such as SLAMF3 and SLAMF5 are not altered. Similarly we observed a small defect in CD1d expression in dnRas DP thymocytes. In contrast, expression of these molecules was not altered in Egr1,2 DKO DP thymocytes. These results suggest that signaling through the SLAM/SAP axis could be impaired in dnRas DP thymocytes, and this could contribute to the positive selection defect. Since the observed defect in expression of some SLAM family members in dnRas DP thymocytes is similar to that observed in cMyb-deficient DP thymocytes, and in these cells there are also defects in the expression of the signaling molecule SAP, and the survival factor BclxL, we decided to analyze expression of these molecules in dnRas mice. In contrast to c-Myb-deficient thymocytes, expression of SAP and BclxL, assessed by intracellular staining, was normal DP thymocytes from dnRas mice. Although it is known that the signaling mechanisms that control iNKT cell development include a component derived from the TCR-CD1d interaction, the pathways that mediate this effect downstream the TCR are not completely understood. In this report we characterize the central contribution of the Ras/MAPK pathway to positive selection of the iNKT cell lineage, similar to its described contribution to conventional ab T cell positive selection. The Ras/MAPK pathway plays a central role during ab T cells positive selection but it is thought to be dispensable for iNKT cell development. In fact, it has been proposed that one of the roles of SLAMs in positive selection of iNKT cells is to block activation of Ras, by inducing recruitment and activation of Ras-GAP. The recent report that thymocytes defective in Egr-2 had a defect in generation of iNKT cells made us reconsider the possible involvement of Ras in this process, since Egr-2 induction downstream the TCR requires activation of both Calcineurin and the Ras/MAPK pathway. Our results clearly show that defects in the Ras/MAPK pathway result in a dramatic blockade in iNKT generation, with accumulation of immature iNKT cell precursors, suggesting a block in the early stages of positive selection. This defect seems mediated by the same downstream Chlormezanone effectors as during conventional ab T cell positive selection, because expression of Egr-2 and Id3 in immature iNKT cell precursors is significantly decreased. It is also possible that the defect in Ras/MAPK activation could interfere with later stages of iNKT cell differentiation that involve proliferation. The Egr family of transcription factors plays an important role in many developmental checkpoints during T cell development. Although in some circumstances they can at least partially compensate for each other, they are not completely redundant. For example, during b-selection Egr-3 is the most important Egr factor. However, compound knockouts show that Egr1 also contributes in this stage. Similarly, during positive selection of conventional ab T cells, both Egr-1 and Egr-2 knockouts show a partial blockade in positive selection. Our results show that, contrary to previous reports, both Egr1 and Egr-2 play a role during iNKT positive selection, although the effect of Egr-1 is subtle, and is only uncovered in mixed bone marrow chimeras, or in the Egr-1, Egr-2 double knockout animals, which have a much more profound block in iNKT generation than Egr-2 knockouts. An interesting HJC0350 difference between the dnRas and the Egr-1, Egr-2 double knockout phenotypes is the observed small defect in the expression of SLAMF1 and SLAMF6 in DP thymocytes from dnRas mice.