Gab2, a newly identified pleckstrin homology domain-containing docking protein, is a major binding protein of SHP-2 tyrosine phosphatase in interleukin (IL)-3-stimulated hematopoietic cells. Its signaling mechanism remains largely unknown. We report here an important regulatory role for Gab2 in beta(1) integrin signaling pathway that mediates hematopoietic cell adhesion and migration. Cross-linking of the beta(1) integrin on Ba/F3 cells induced rapid tyrosine phosphorylation of Gab2 and its association with Syk kinase, SHP-2 phosphatase, and the p85 subunit of phosphatidylinositol (PI)-3 kinase. In addition, Gab2 was also constitutively associated with SHP-1 phosphatase via its C-terminal Src homology 2 domain. Overexpression of the pleckstrin homology domain or a mutant Gab2 molecule lacking SHP-2 binding sites resulted in significant reductions in Ba/F3 cell adhesion and migration. Biochemical analyses revealed that enforced expression of Gab2 mutant molecules dramatically reduced beta(1)-integrin ligation-triggered PI3 kinase activation, whereas Erk kinase activation remained unaltered. Furthermore, transduction of primary hematopoietic progenitor cells from viable motheaten mice with these mutant Gab2 molecules also significantly ameliorated their enhanced migration capacity associated with the SHP1 gene mutation. Taken together, these results suggest an important signaling role for Gab2 in regulating hematopoietic cell adhesion and migration.