Higher plants have a vascular system that ensures the distribution of water and energy in form of sugars. The latter are transported in sieve elements, specialized tube-like cells in the phloem. While several regulators of sieve element differentiation have been described, many remain unknown. Furthermore, the temporal cascade of differentiation regulation remains unclear due to limited resolution in published data. In this study, using fluorescent reporters limited to specific developmental stages of protophloem sieve elements (PSE) combined with fluorescence activated cell sorting, new phloem specific transcription factors (TFs) were identified. To increase the temporal resolution, single cell transcriptomics, based on the same reporter lines, were used. Analysis of the resulting data set allowed ordering of 758 single cell transcriptomes, representing the 22-cell developmental trajectory of PSE cells in the root. This order was in good accordance with the known expression pattern of previously described regulators and allowed the precise temporal description of newly identified genes. In addition, four distinct developmental stages along the PSE differentiation trajectory could be identified, highlighting the drastic changes from transit amplifying cells to early and early to late differentiating cells. In addition, a group of PSE expressed DOF transcription factors, called PEARs, had been found previously. In this work, the redundancy of these DOFs controlling the proliferation of procambial cells in a non-cell-autonomous manner, was resolved, and a combinatorial sextuple mutant, lacking nearly all periclinal cell divisions in the vasculature, was identified. Furthermore, this mutant displayed a PSE differentiation phenotype. The expression of the previously newly identified phloem specific transcription factors was investigated in this mutant background. These analyses placed the PEARs on top of the known TFs cascade controlling PSE differentiation. Taken together, this work elucidates temporal aspects and new regulators of PSE differentiation as well as the role of these cells in root meristem formation.