In classical descriptions of leukocyte chemokine signaling, Src family kinases are thought to function in a positive fashion by coupling receptor associated Gα subunits to downstream mitogen activated protein (MAP) kinase activation. However, neutrophils derived from hck^−/−fgr^−/− mice and dendritic cells (DCs) from fgr^−/− animals manifested significantly higher intracellular signaling (Ca2+ flux, MAP kinase activation, actin polymerization) and functional responses (chemotaxis in vitro and migration in vivo) to a number of different chemokines. These kinases may mediate their effect through the inhibitory receptor PIR-B since neutrophils and DCs from pir-b^−/− mice were also hyperresponsive to chemokine stimulation. In wild-type (wt) cells dephosphorylation of PIR-B was associated with maximal chemokine signaling, whereas in hck^−/−fgr^−/− cells PIR-B was unphosphorylated. These data support a model in which the Src family kinases Hck and Fgr function as negative regulators of myeloid cell chemokine signaling by maintaining the tonic phosphorylation of PIR-B.