Despite the loss of proximal TCR-dependent signaling events, downstream T cell responses are paradoxically augmented in T cells with siRNA-mediated Lck knockdown (Methi et al., J. Immunol. 2005. 175: 7398–7406). This indicates that alternative Lck-independent pathways of T cell activation exist or that low levels of Lck elicit other signals than normal T cell activation. Here we report the recruitment of Grb2-SOS1 to CD3ζ of the TCR complex after prolonged anti-CD3 (OKT3) stimulation in T cells with Lck knockdown. Grb2 bound to incompletely phosphorylated ITAM1 with the pY-Y configuration in a solid-phase assay, but was excluded by ZAP-70 in the doubly phosphorylated pY-pY conformation. Ras and ERK1/2 activation was augmented after prolonged stimulation in T cells with Lck knockdown compared to control, leading to increased activation of the proximal IL-2 promoter (NFAT-AP-1). Finally, the phosphorylation of Ras-GAP was strongly suppressed in Lck knockdown cells, indicating that a Ras negative feedback mechanism is dependent on Lck.