Abstract: Mammalian T-cell responses require synergism between the first signal and co-stimulatory signal. However, whether and how dual signaling regulates the T-cell response in early vertebrates remains unknown. In the present study, we discovered that the Nile tilapia (Oreochromis niloticus) encodes key components of the LAT signalosome, namely, LAT, ITK, GRB2, VAV1, SLP-76, GADS, and PLC-γ1. These components are evolutionarily conserved, and CD3ε mAb-induced T-cell activation markedly increased their expression. Additionally, at least ITK, GRB2, and VAV1 were found to interact with LAT for signalosome formation. Downstream of the first signal, the NF-κB, MAPK/ERK, and PI3K-AKT pathways were activated upon CD3ε mAb stimulation. Furthermore, treatment of lymphocytes with CD28 mAbs triggered the AKT-mTORC1 pathway downstream of the co-stimulatory signal. Combined CD3ε and CD28 mAb stimulation enhanced ERK1/2 and S6 phosphorylation and elevated NFAT1, c-Fos, IL-2, CD122, and CD44 expression, thereby signifying T-cell activation. Moreover, rather than relying on the first or co-stimulatory signal alone, both signals were required for T-cell proliferation. Full T-cell activation was accompanied by marked apoptosis and cytotoxic responses. These findings suggest that tilapia relies on dual signaling to maintain an optimal T-cell response, providing a novel perspective for understanding the evolution of the adaptive immune system.