It has been demonstrated, that gammadelta T cells play an important role in the development of immune responses to many pathogens. gammadelta T cells play a role in the clearance of viral and microbiological infections, anti-tumor responses, but also in autoimmune diseases. Many different protocols for the isolation and cultivation of gammadelta T cells can be found in the literature. Here we compare three common cultivation protocols for gammadelta T cells derived from peripheral blood with a newly developed protocol depending on SLAM (Signaling Lymphocyte Activation Molecule) stimulation. We demonstrate that the cultivation protocol chosen to raise gammadelta T cells has direct impact on the resulting gammadelta T cell phenotype. We show differences in gammadelta TCR composition, memory phenotype formation, CD8 receptor expression and the expression of NK cell markers depending on the stimulation protocol used. As such, the cultivation protocol chosen for a series of experiments might have significant impact on the outcome of the experiments and should be considered carefully.