Recruitment of polymorphonuclear neutrophils (PMN) to sites of acute inflammation critically depends on β2 integrins (CD11/CD18). Recently, the mammalian actin-binding protein 1 (mAbp1) was identified as an important adaptor protein regulating PMN trafficking downstream of β2 integrins. Here, we show that mAbp1 constitutively co-immunoprecipitated with hematopoietic progenitor kinase 1 (HPK1) in neutrophil-like differentiated HL-60 (dHL-60) cells. HPK1 was enriched at the lamellipodium of polarized dHL-60 cells, where it co-localized with mAbp1 and actin. Functional analysis of PMN from HPK1 deficient mice showed that HPK1 was critical for CXCL1-induced lymphocyte function-associated antigen 1 (LFA-1)-mediated PMN adhesion to ICAM-1 under flow conditions. Accordingly, CXCL1-mediated induction of high affinity LFA-1 required HPK1, while macrophage antigen 1 (Mac-1) affinity regulation was independent of HPK1. Intravital microscopy of the mouse cremaster muscle confirmed the defect of CXCL1-induced leukocyte adhesion in HPK1 deficient mice. Furthermore, β2 integrin-mediated post-adhesion processes namely adhesion strengthening, spreading, and directed mechanotactic crawling of PMN under flow conditions involved HPK1 in vitro and in vivo. Upon intrascrotal administration of tumor necrosis factor α (TNFα), PMN adhesion and extravasation were severely compromised in HPK1 deficient mice. In summary, our results indicate that HPK1 is critically involved in LFA-1-mediated PMN trafficking during acute inflammation.