Although the evolutionary importance of spontaneous mutation is evident, its contribution to the evolution of ecological specificity remains unclear, because the environmental sensitivity of effects of new mutations has received little empirical attention. To address this issue, we report a greenhouse in which we grew plants from 20 mutation-accumulation (MA) lines, advanced by selfing and single-seed descent from a single common founder to generation 17, as well as plants from five lines representing the founder, in high and low nutrient conditions. We examined 11 traits throughout life history, including germination, survivorship, bolting date, flowering date, leaf number, leaf size, early and late height, mean fruit size, total seed weight, and reproductive biomass. Comparison of trait means between the two generations did not support the commonly held view that new mutations affecting fitness in these MA lines are strongly biased toward deleterious effects. We detected significant variance among MA lines for one fitness component, mean fruit size, but we did not detect a significant contribution of mutations accumulated in these MA lines to genotype by environment interaction (GEI). These results suggest that other evolutionary mechanisms play a more important role than spontaneous mutation alone in establishing the GEI found for wild collections and lab accessions of Arabidopsis thaliana in previous studies.