One of the factors that can impact human embryonic stem cell expansion in stirred microcarrier culture reactors is mechanical stress caused by agitation. Therefore, we have investigated the effects of agitation on human embryonic stem cell growth and expression of pluripotent markers. Agitation of HES-2 cell line in microcarrier cultures in stirred spinner and agitated six-well plates did not affect expression of pluripotent markers, cell viability, and cell doubling times even after seven passages. However, HES-3 cell line was found to be shear sensitive, showing downregulation of three pluripotent markers Oct-4, mAb 84, and Tra-1-60, and lower cell densities in agitated as compared with static cultures, even after one passage. Cell viability was unaffected. The HES-3-agitated cultures showed increased expression of genes and proteins of the three germ layers. We were unable to prevent loss of pluripotent markers or restore doubling times in agitated HES-3 microcarrier cultures by addition of five different known cell protective polymers. In addition, the human induced pluripotent cell line IMR90 was also shown to differentiate in agitated conditions. These results indicate that the effect of agitation on cell growth and differentiation is cell line specific. We assume that the changes in the growth and differentiation of the agitation-sensitive (HES-3) cell line do not result from the effect of shear stress directly on cell viability, but rather by signaling effects that influence the cells to differentiate resulting in slower growth.