A comprehensive analysis of T cell activation markers in chicken is lacking. Kinetics of T cell activation markers (CD25, CD28, CD5, MHC-II, CD44, and CD45) in response to in vitro stimulation of peripheral blood mononuclear cells with concanavalin A (Con A) were evaluated between two chicken lines selected for high and low levels of mannose-binding lectin in serum (L10H and L10L, respectively) by flow cytometry. L10H chickens showed a stronger response to Con A based on the frequency of T cell blasts in both the CD4+ and CD8+ compartment. The majority of the proliferating CD4+ and CD8+ T cells expressed CD25. Proliferating T cells were seen both in the CD4+ MHC-II+/- and CD8+ MHC-II+/- population. For both CD4+ and CD8+ T cells, frequencies of CD25+ and MHC-II+ T cells were increased 24 h after stimulation. CD28+ frequencies were only increased on CD8+ T cells 48 h after stimulation. An increase in the relative surface expression based on mean fluorescence intensity (MFI) upon activation was observed for most markers except CD5. For CD4+ T cells, CD28 expression increased 24 h after stimulation whereas MHC-II expression increased after 48 h. For CD8+ T cells, a tendency toward an increase in CD25 expression was observed. CD28 expression started to increase 24 h after stimulation and only a transient peak in MHC-II expression on CD8+ T cells was observed after 24 h. CD44 and CD45 expressed on CD4+ and CD8+ T cells increased 24-72 h after stimulation. In summary, the frequency of CD25+ and MHC-II+ T cells were shown to be early markers (24 h) for in vitro activation of both CD4+ and CD8+ T cells. Frequency of CD28+ T cells was a later marker (48 h) and only for CD8+ T cells. Surface expression of all markers (MFI) increased permanently or transiently upon activation except for CD5.