Engagement of neutrophils by E-selectin results in integrin activation. Here, we investigated primary mouse neutrophils in whole blood by using intravital microscopy and autoperfused flow chambers. Slow rolling on E-selectin coimmobilized with intercellular adhesion molecule-1 (ICAM-1) required P-selectin glycoprotein ligand (PSGL)-1, was dependent on α_Lβ₂ integrin (LFA-1), and required continuous E-selectin engagement. Slow rolling was abolished by pharmacological blockade of spleen tyrosine kinase (Syk) and was absent in Syk^−/− bone-marrow chimeric mice. Treatment with tumor necrosis factor-α lowered rolling velocity further and induced CXC chemokine ligand-1 (CXCL1) and CXC chemokine receptor-2 (CXCR2)-dependent leukocyte arrest on E-selectin and ICAM-1. Arrest but not rolling was blocked by an allosteric inhibitor of LFA-1 activation. Neutrophil recruitment in a thioglycollate-induced peritonitis model was almost completely inhibited in Selplg^−/− mice or Syk^−/− bone-marrow chimeras treated with pertussis toxin. This identifies a second neutrophil-activation pathway that is as important as activation through G protein-coupled receptors (GPCRs).