Serum Response Factor (SRF) recruits members of two families of signal-regulated coactivators, the ERK-regulated TCFs and the actin-regulated MRTFs, to its target genes through its DNA-binding domain. Whether coactivator association is required for SRF function in vivo, and whether particular SRF functions reflect specific coupling to one or other signal pathway has remained largely unexplored. We show that SRF is essential for thymocyte positive selection and thymic Treg and NKT cell development, but dispensible for early thymocyte development and negative selection. Expression of wildtype SRF, or mutants lacking the N-terminal phosphorylation sites or C-terminal transcriptional activation domain, restores positive selection in SRF null thymocytes. In contrast, SRF.V194E, which cannot recruit TCF- or MRTF-family members, is inactive although it is recruited to target genes. Fusion of a TCF C-terminal activation domain to SRF.V194E effectively restores ERK-dependent SP thymocyte development. The resulting SP thymocytes exhibit normal surface marker expression and proliferation following TCR crosslinking. Thus ERK signalling through the TCF pathway to SRF is necessary and sufficient for SRF function in thymocyte positive selection.