Cells have a regulatory mechanism known as heat shock (HS) response, which induces the expression of HS genes and proteins in response to heat and other cellular stresses. Exposure to moderate HS results in beneficial effects, such as thermotolerance and promotes survival, whereas excessive HS causes cell death. The effect of HS on cells depends on both exogenous factors, including the temperature and duration of heat application, and endogenous factors, such as the degree of cell differentiation. Neural stem cells (NSCs) can self-renew and differentiate into neurons and glial cells, but the changes in the HS response of symmetrically proliferating NSCs in culture are unclear. We evaluated the HS response of homogeneous proliferating NSCs derived from mouse embryonic stem cells during the proliferative phase and its effect on survival and cell death in vitro. The number of adherent cells and the expression ratios of HS protein (Hsp)40 and Hsp70 genes after exposure to HS for 20 min at temperatures above 43°C significantly increased with the extension of the culture period before exposure to HS. In contrast, caspase activity was significantly decreased by extension of the culture period before exposure to HS and suppressed the decrease in cell viability. These results suggest that the culture period before HS remarkably affects the HS response, influencing the expression of HS genes and cell survival of proliferating NSCs in culture.