Single somatic plant cells, after wall digestion to become a protoplast, has the ability to become a new fertile plant. The regeneration process is commonly divided into two steps, callus formation and shoot formation. Protoplast regeneration is not only a striking example of cell fate switch, but also has potential in agrobiotechnology, including transformation and gene editing. However, regeneration rate is usually very low, hampering broad application of the technique. Starting from Arabidopsis mesophyll cells, we found that only a small portion (~3%) of regenerated calli form shoots. Single cell transcriptomic analysis in combination with live imaging showed that callus fate is heterogeneous and changes during culture. We found that callus can be broadly divided into two categories. The first cell group shares similarity with vascular precures and eventually lignify. The other group cell group include shoot precursors and initially express the shoot meristem marker gene WUSCHEL (WUS). WUS inhibits vascular cell fate, and vice versa. Together, we show that single cell derived callus can still have variable cell fate, which is largely noise driven. Modulating cell fate provide a means to boost regenerate efficiency.