Plant somatic cells can be reprogrammed to form a pluripotent cell mass, known as callus, from which new organs can grow via de novo organogenesis. Here, we show that pluripotency acquisition during callus formation is transient, regardless of explant origin. Using single-cell transcriptome analysis of pluripotent and nonpluripotent calli, we discovered that stem cell activity, conferred by the WUSCHEL-RELATED HOMEOBOX5 (WOX5)–TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) module, decreases upon prolonged callus incubation on CIM. This loss of stem cell activity is associated with the activation of BEARSKIN 1 (BRN1), which promotes differentiation into lateral root cap (LRC)-like cells. This transition is accompanied by the depletion of epidermis-like cells accumulating very long-chain fatty acids that are potentially linked to the maintenance of pluripotency. Furthermore, reactive oxygen species (ROS) accumulation, hypoxia response, and salicylic acid (SA) biosynthesis are activated in mature LRC-like cells, collectively contributing to the loss of pluripotency. We propose that callus formation follows a continuous developmental program, in which stem cell activity is transiently acquired and subsequently lost, explaining the transient nature of cellular pluripotency in callus.