Despite its genetic homogeneity, a clonal population can display marked heterogeneity in various aspects, such as growth rate and stress resistance. Such heterogeneity may have important clinical implications in tumor biology and microbial pathogenesis. As a model eukaryote and opportunistic pathogen, the budding yeast Saccharomyces cerevisiae provides an excellent model for studying non-genetic heterogeneity. Isogenic yeast cells, cultivated under benign conditions, divide at different rates. Slow-growing cells disproportionately survive acute heat shock and have high expression of a stress-protective factor, TSL1. Knocking out TSL1 significantly reduces survival of acute heat shock but has negligible effect on growth. The molecular mechanisms that generate non-genetic variation in yeast-cell growth rate and stress resistance have not been characterized. We identified the paralogous transcription factors MSN2 and MSN4, and the upstream protein kinase A (PKA) pathway, as candidate regulators of this heterogeneity. MSN2 and MSN4 regulate the general stress response. PKA regulates their phosphorylation status, which determines their nuclear import and export rates. Although there have been extensive studies on the function and subcellular localization dynamics of MSN2 and MSN4 under stress, the significance of MSN2 and MSN4 under benign conditions has rarely been explored. We use high-throughput time-lapse microscopy to measure growth and gene expression in microcolonies founded by individual cells. The negative correlation between single-cell growth rate and TSL1 expression level is abolished in msn2 mutants but not msn4 mutants, which suggests only MSN2 is necessary in regulating heterogeneity in TSL1 expression and stress resistance. The population growth-rate distributions of msn2, msn4 and msn2 msn4 double mutants all show similar, significant reduction of slow growers compared with wildtype. These results suggest that MSN2 and MSN4 are each necessary in regulating slow growers. The proportion of slow growers also decreases significantly in mutants lacking subunits of the PKA complex, TPK1 or TPK2, and the decrease is even stronger than in msn2 mutants. Therefore, PKA regulates slow growers, but not solely through MSN2 and MSN4. Monitoring of MSN2 intracellular localization reveals that heterogeneity likely derives in part from its nuclear dwell time: MSN2 enters the nucleus more frequently and stays there longer in slow-growing cells, presumably leading to differential expression of target genes and thereby different growth and stress-resistance states.