A substantial lack of information is recognized on the features underlying the variable susceptibility to amyloid aggregate toxicity of cells with different phenotypes. Recently, we showed that different cell types are variously affected by early aggregates of a prokaryotic hydrogenase domain (HypF-N). In the present study we investigated whether differentiation affects cell susceptibility to amyloid injury using a human neurotypic SH-SY5Y cell differentiation model. We found that retinoic acid-differentiated cells were significantly more resistant against Aβ1-40, Aβ1-42 and HypF-N prefibrillar aggregate toxicity respect to undifferentiated cells treated similarly. Earlier and sharper increases in cytosolic Ca2+ and ROS with marked lipid peroxidation and mitochondrial dysfunction were also detected in exposed undifferentiated cells resulting in apoptosis activation. The reduced vulnerability of differentiated cells matched a more efficient Ca2+-ATPase equipment and a higher total antioxidant capacity. Finally, increasing the content of membrane cholesterol resulted in a remarkable reduction of vulnerability and ability to bind the aggregates in either undifferentiated and differentiated cells.