De novo shoot regeneration in tissue culture experiences at least two phases. Explants are first cultured on auxin rich callus-inducing medium (CIM) to produce a group of pluripotent cells termed callus; the callus is then transferred to cytokinin rich shoot-inducing medium (SIM) to promote the formation of shoot progenitor cells, from which adventitious shoots may differentiate. Here, we show that the Arabidopsis thaliana transcription factor gene LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) is involved in pluripotency acquisition in callus cells. LBD16, which is activated by WUSCHEL RELATED HOMEOBOX11 (WOX11), is specifically expressed in the newly formed callus on CIM and its expression decreases quickly when callus is moved to SIM. Blocking the WOX11-LBD16 pathway results in the loss of pluripotency in callus cultured on CIM, leading to shooting defects on SIM. Further analysis showed that LBD16 may function in the establishment of the root primordium-like identity in the newly formed callus, indicating that the root primordium-like identity is the cellular nature of pluripotency in callus cells. Additionally, LBD16 promotes cell division during callus initiation. Our study clarified that the WOX11-LBD16 pathway promotes pluripotency acquisition in callus cells.