Flavonoids have broad cross-kingdom biological activity. In Arabidopsis, flavonoid accumulation in specific tissues, notably the root elongation zone and root/shoot junction modulate auxin transport, affect root gravitropism, and influence overall plant architecture. The relative contribution made by aglycones and their glycosides remains undetermined, and the longer-term phenotypic effects of altered flavonoid accumulation are not fully assessed. We tested Arabidopsis thaliana mutants that accumulate different flavonoids to determine which flavonoids were causing these affects. Tandem mass spectrometry and in situ fluorescence localisation were used to determine the in vivo levels of aglycones in specific tissues of 11 transparent testa mutants. We measured rootward and shootward auxin transport, gravitropic responses, and identified the long-term changes to root and shoot architecture. Unexpected aglycone species accumulated in vivo in several flavonoid-pathway mutants, and lower aglycone levels occurred in transcription factor mutants. Mutants accumulating more quercetin and quercetin-glycosides changed the greatest in auxin transport, gravitropism, and aerial tissue growth. Early flavonoid-pathway mutants showed aberrant lateral root initiation patterns including clustered lateral root initiations at a single site. Transcription factor mutants had multiple phenotypes including shallow root systems. These results confirm that aglycones are present at very low levels, show that lateral root initiation is perturbed in early flavonoid-pathway mutants, and indicate that altered flavonoid accumulation affects multiple aspects of plant architecture.