A ntagonists to α4 integrin show promise for several autoimmune and inflammatory diseases but may exhibit mechanism-based toxicities. We tested the capacity of blockade of α4 integrin signaling to perturb functions involved in inflammation, while limiting potential adverse effects. We generated and characterized mice bearing a Y991A mutation in α4 integrin [α4(Y991A) mice], which blocks paxillin binding and inhibits α4 integrin signals that support leukocyte migration. In contrast to the embryonic-lethal phenotype of α4 integrin–null mice, mice bearing the α4(Y991A) mutation were viable and fertile; however, they exhibited defective recruitment of mononuclear leukocytes into thioglycollate-induced peritonitis. α4 Integrins are essential for definitive hematopoiesis; however, the α4(Y991A) mice had intact lymphohematopoiesis and, with the exception of reduced Peyer’s patches, normal architecture and cellularity of secondary lymphoid tissues. We conclude that interference with α4 integrin signaling can selectively impair mononuclear leukocyte recruitment to sites of inflammation while sparing vital functions of α4 integrins in development and hematopoiesis.