Avian γδ T lymphocytes still represent an enigmatic cell population not only regarding their functions but also their activation requirements and subset differentiation. To find a suitable method for non-specific stimulation and multiplication of CD8α-characterised γδ T-cell subsets in peripheral blood lymphocyte (PBL) cultures, PBLs of four different chicken lines (WLA, BLA, R11, L68) were treated with a range of commonly used non-specific reagents (PMA, PHA, ConA) and interleukins (IL-2, IL-12, IL-15), and the CD8α−, CD8αhiβ+ and CD8ααhi+ γδ T-cell subsets examined in relation to activation (CD25 expression) and proliferation by flow cytometry. The culture of avian PBLs with PMA led to an increase of CD25-expression intensity, whereas IL-2 induced proliferation of γδ T-cells. The combinational application of IL-2 plus PMA functioned synergistically and resulted in significantly enhanced numbers of CD25+ cells, with simultaneous significant increase of the CD25-expression intensity and proliferation in all γδ T-cell subsets of all chicken lines. The four chicken lines revealed only sporadically differences with frequently highest proliferation rates in PBLs of WLA. In conclusion, PMA in combination with IL-2 is the best suitable additive for avian PBL cultures in order to multiply, activate and maintain CD8α-characterised γδ T-cell subsets of different chicken lines.