We report experiments on the connection between nucleolar stress and cell cycle progression, using HeLa cells engineered with the fluorescent ubiquitinylation-based cell cycle indicator. Nucleolar stress elicited by brief exposure of cells to a low concentration of actinomycin D that selectively inhibits rRNA synthesis had no effect on traverse of G1 or S, but stalled cells in very late interphase. Additional experiments revealed that a switch occurs during a specific temporal window during nucleolar stress and that the subsequent cell cycle arrest is not triggered simply by the stress-induced decline in the synthesis of rRNA or by a ribosome starvation phenomenon. Further experiments revealed that this nucleolus stress-induced cell cycle arrest involves the action of a G2 checkpoint mediated by the ataxia telangiectasia and Rad3-related protein (ATR)–checkpoint kinase 1 (Chk1) pathway. Based on analysis of the cell cycle stages at which this nucleolar stress effect is put into action, to become manifest later, our results demonstrate a feedforward mechanism that leads to G2 arrest and identify ATR and Chk1 as molecular agents of the requisite checkpoint.