Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 serine/threonine kinase super family. We recently reported that the immunosuppressive eicosanoid, prostaglandin E2 (PGE 2), is capable of activating HPK1 in T cells. In this report, we demonstrate that unlike the TCR-induced activation of HPK1 kinase activity, the induction of HPK1 catalytic activity by PGE2 does not require the presence of phosphotyrosine-based signaling molecules such as Lck, ZAP-70, SLP-76, and Lat. Nor does the PGE2-induced HPK1 activation require the intermolecular interaction between its proline-rich regions and the SH3 domain-containing adaptor proteins, as required by the signaling from the TCR to HPK1. Instead, our study reveals that PGE2 signal to HPK1 via a 3′-5′-cyclic adenosine monophosphate-regulated, PKA-dependent pathway. Consistent with this observation, changing the serine 171 residue that forms the optimal PKA phosphorylation site within the "activation loop" of HPK1 to alanine completely prevents this mutant from responding to PGE2-generated stimulation signals. Moreover, the inability of HPK1 to respond to PGE2 stimulation in PKA-deficient S49 cells further supports the importance of PKA in this signaling pathway. We speculate that this unique signaling pathway enables PGE2 signals to engage a proven negative regulator of TCR signal transduction pathway and uses it to inhibit T cell activation.