The role of integrin-mediated signaling events in T cell function remains incompletely characterized. We report here that α4β1 integrin stimulation of H9 T cells and normal human T cell blasts results in rapid and transient tyrosine phosphorylation of the adapter protein, SH2 domain- containing 76-kDa protein (SLP-76)-associated phosphoprotein of 130 kDa (SLAP-130)/FYB at levels comparable to those observed following TCR stimulation. Stimulation of T cells via the α4β1 integrin enhances the association of tyrosine phosphorylated SLAP-130/FYB with the SH2 domain of the src tyrosine kinase p59(fyn). Activation of normal T cells, but not H9 T cells, via α4β1 leads to tyrosine phosphorylation of SLP-76 as well as SLAP-130/FYB. Overexpression of SLAP-130/FYB in normal T cells enhances T cell migration through fibronectin-coated filters in response to the chemokine stromal cell-derived factor (SDF)-1α. These results identify SLAP- 130/FYB as a new tyrosine phosphorylated substrate in β1 integrin signaling and suggest a novel function for SLAP-130/FYB in regulating T lymphocyte motility.