Postinfiltration air temperature is known to affect the accumulation of recombinant protein in Agrobacterium-mediated transient expression in Nicotiana benthamiana plants, including the number of days needed to reach maximum content and the rate of reduction thereafter. This study aimed to clarify whether the transcript levels of the transgenes and those of plant stress response markers (i.e., hypersensitive response and endoplasmic reticulum [ER] stress) could be primary determinants of the accumulation of recombinant influenza hemagglutinin (HA) at 21 or 26°C. We found no correlation between the transgene expression levels (HA, RdRP, and MP) and the number of days needed to reach the maximum HA protein content at both temperatures. Regardless of the accumulation compartment, HA protein content peaked earlier at 26°C than at 21°C. The rapid reduction of HA content after reaching the maximum, observed only in the ER at 26°C, correlated with severe necrosis and high transcript levels of two representative ER stress markers, bZIP60 and BiP. This correlation suggests that high postinfiltration air temperature affects HA accumulation primarily through ER stress, a key factor in the rapid reduction of HA content after the peak.