Characterizing the biological effects of flattening filter-free (FFF) X-ray beams from linear accelerators is of importance, due to their increasing clinical availability. The purpose of this work is to determine whether in vitro cell survival is affected by the higher dose-per-pulse present in FFF beams in comparison with flattened X-ray beams. A Varian TrueBeam(®) linear accelerator was used to irradiate the T98G, V79-4 and U87-MG cell lines with a single fraction of 5 Gy or 10 Gy doses of X-rays. Beams with energies of 6 MegaVolt (MV), 6 MV FFF and 10 MV FFF were used, with doses-per-pulse as measured at the monitor chamber of 0.28, 0.78 and 1.31 mGy/pulse for 6 MV, 6 MV FFF and 10 MV FFF, respectively. The dose delivered to each Petri dish was verified by means of ionization chamber measurements. No statistically significant effects on survival fraction were observed for any of the cell lines considered, either as a function of dose-per-pulse, average dose rate or total dose delivered. Biological effects of higher instantaneous rates should not be excluded on the basis of in vitro experimental results such as the ones presented in this work. The next step toward an assessment of the biological impact of FFF beams will require in vivo studies.