In plant tissue culture, callus is induced by a high auxin concentration. Among the three cell layers (the outer, middle, and inner cell layers) of callus, pluripotency acquisition in the middle cell layer is critical for the potential ability of callus to regenerate organs. Here, we reveal the developmental trajectory of middle cell layer initiation and maintenance in callus originating from Arabidopsis thaliana hypocotyls. The S phase in cell cycle is essential for the expression of quiescent center (QC)-related SCARECROW (SCR), PLETHORA1 (PLT1), and WUSCHEL-RELATED HOMEOBOX5 (WOX5) genes during the division of callus founder cells to initiate the callus primordium. After callus initiation, SHOOT-ROOT (SHR) proteins move from the inner to the middle cell layer and act together with SCR to promote the expression of PLT1 and WOX5. WOX5 represses the expression of VASCULAR-RELATED NAC-DOMAIN (VND) genes, thereby preventing callus from differentiating into the xylem. PLT1/2 directly activate JACKDAW (JKD) for pluripotency acquisition in the middle cell layer. We hypothesize that the middle cell layer could have pluripotent stem cell activity and its establishment requires the QC-related SCR-SHR-WOX5-PLT1/2-JKD gene network.