Auxin is important for lateral root (LR) initiation and subsequent LR primordium development. However, the roles of tissue-specific auxin signaling in these processes are poorly understood. We analyzed transgenic Arabidopsis plants expressing the stabilized mutant INDOLE-3 ACETIC ACID 14 (IAA14)/SOLITARY-ROOT (mIAA14) protein as a repressor of the auxin response factors (ARFs), under the control of tissue-specific promoters. We showed that plants expressing the mIAA14-glucocorticoid receptor (GR) fusion protein under the control of the native IAA14 promoter had the solitary-root/iaa14 mutant phenotypes, including the lack of LR formation under dexamethasone (Dex) treatment, indicating that mIAA14-GR is functional in the presence of Dex. We then demonstrated that expression of mIAA14-GR under the control of the stele-specific SHORT-ROOT promoter suppressed LR formation, and showed that mIAA14-GR expression in the protoxylem-adjacent pericycle also blocked LR formation, indicating that the normal auxin response mediated by auxin/indole-3 acetic acid (Aux/IAA) signaling in the protoxylem pericycle is necessary for LR formation. In addition, we demonstrated that expression of mIAA14-GR under either the ARF7 or the ARF19 promoter also suppressed LR formation as in the arf7 arf19 double mutants, and that IAA14 interacted with ARF7 and ARF19 in yeasts. These results strongly suggest that mIAA14-GR directly inactivates ARF7/ARF19 functions, thereby blocking LR formation. Post-embryonic expression of mIAA14-GR under the SCARECROW promoter, which is expressed in the specific cell lineage during LR primordium formation, caused disorganized LR development. This indicates that normal auxin signaling in LR primordia, which involves the unknown ARFs and Aux/IAAs, is necessary for the establishment of LR primordium organization. Thus, our data show that tissue-specific expression of a stabilized Aux/IAA protein allows analysis of tissue-specific auxin responses in LR development by inactivating ARF functions.