Two GRAS family transcription factors, SHORT-ROOT (SHR) and SCARECROW (SCR), are required for ground tissue and quiescent center formation in Arabidopsis roots. The action of SHR and SCR is regulated by two INDETERMINATE DOMAIN (IDD) family proteins, JACKDAW (JKD) and MAGPIE (MGP). Although the reciprocal interaction of these transcription factors is considered to be involved in the modulation of SHR and SCR action by JKD and MGP, the underlying mechanism remains unclear. In this study, we use a transient assay with Arabidopsis culture cells to show that the physical interaction of these transcription factors modulate their transcriptional activity. Transient expression of LUC reporter genes with the proximal sequences upstream from the ATG codon of SCR and MGP in protoplasts were activated by JKD. Moreover, promoter activities were enhanced further by the addition of SHR and SCR to JKD, but not by the combination of SHR and SCR in the absence of JKD. Yeast one-hybrid analysis showed that JKD binds to the SCR and MGP promoter sequences, indicating the existence of another binding sequences of JKD different from the previously determined IDD binding sequence. Our findings suggest that JKD directly regulates SCR and MGP expression in cooperation with SHR, SCR and MGP.