Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an attaching and effacing pathogen that upon attachment to host cells, induce characteristic attaching and effacing lesions and formation of F-actin rich pedestals beneath sites of bacterial attachment. EHEC harbors a Type III secretion system through which it delivers dozens of effectors into the host cell. The two secreted effectors critical for EHEC-mediated actin pedestal formation are the translocated intimin receptor (Tir) and EspF_U. EspF_U consists of an N-terminal secretion signal and a C-terminus containing six tandem 47-residue proline-rich repeats, each of which can bind and activate the actin nucleation promoting factor N-WASP. Structural and functional analyses described here have identified the mechanism of N-WASP activation by EspF_U and the minimal domains and specific residues required for this activity. While EspF_U and Tir are the only bacterial effectors required for F-actin pedestal formation, recruitment of EspF_U to Tir is mediated by an unidentified putative host factor. To identify the host factor responsible for linking these two effectors, a combination of in vitro and functional assays were used to identify the host factor, IRTKS and the residues required for these interactions were defined. Further, the presence of at least two 47-residue repeats in all characterized clinical isolates of canonical EHEC strains led us to address the minimal requirements for EspF_U functional domains to promote recruitment to Tir and N-WASP activation. Here we show that two proline-rich elements of EspF_U are required for recruitment of EspF_U by IRTKS to sites of bacterial attachment. Furthermore, once artificially clustered at the membrane, a single N-WASP binding element of EspF_U can induce actin pedestal formation.