Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related serine/threonine protein kinase, is a hematopoietic-specific upstream activator of the c-Jun N-terminal kinase. Here, we provide evidence to demonstrate the involvement of HPK1 in T cell receptor (TCR) signaling. HPK1 was activated and tyrosine-phosphorylated with similar kinetics following TCR/CD3 or pervanadate stimulation. Co-expression of protein-tyrosine kinases, Lck and Zap70, with HPK1 led to HPK1 activation and tyrosine phosphorylation in transfected mammalian cells. Upon TCR/CD3 stimulation, HPK1 formed inducible complexes with the adapters Nck and Crk with different kinetics, whereas it constitutively interacted with the adapters Grb2 and CrkL in Jurkat T cells. Interestingly, HPK1 also inducibly associated with linker for activation of T cells (LAT) through its prolinerich motif and translocated into glycolipid-enriched microdomains (also called lipid rafts) following TCR/CD3 stimulation, suggesting a critical role for LAT in the regulation of HPK1. Together, these results identify HPK1 as a new component of TCR signaling. T cell-specific signaling molecules Lck, Zap70, and LAT play roles in the regulation of HPK1 during TCR signaling. Differential complex formation between HPK1 and adapters highlights the possible involvement of HPK1 in multiple signaling pathways in T cells.