Developmental biology focuses on studying how cells, tissues, and entire organs grow and develop. At the foundational level of this developmental process lies the identification of the emergent properties that allow the passage of stem cells to differentiated cells. For plant cells, which are immobile, positional information rather than lineage becomes crucial for determining cell identity and differentiation. The Arabidopsis root apical meristem (RAM) serves as a relatively simply organized, traceable model to study the morphological, molecular, and physiological aspects of stem cell maintenance and differentiation. All root cells originate from the stem cell niche (SCN) in the center of the RAM. The SCN can be separated into two distinct groups of cells: the relatively mitotically inactive quiescent center (QC) and the surrounding initial cells, which have the potential for differentiation. The QC plays a central role in regulating stem cell initial maintenance, which is further discussed in this review. From the SCN shootwards, the root is made up of concentric rings of individual cell files (epidermis, cortex, endodermis, and pericycle) surrounding a central vasculature bundle (phloem and xylem). From the SCN downwards and laterally, we find the columella and lateral root cap (Figure 1a). Thus, along the longitudinal axis of the root, cells divide, transition to expansion, and differentiate into specialized cells. As such, sampling the root into the longitudinal (and/or radial) axis allows dissecting all the developmental stages from stem cells to differentiated cells (in a cell type–specific manner). In this review, we focus on the different levels of regulations, including transcriptional and post-translational regulation, protein–protein interactions, and mobile signals, within the SCN to coordinate cell differentiation and subsequently instruct tissue patterning and overall root development. We specifically focus on the regulatory interactions governing the QC and the initial cells directly adjacent to it, the columella initials (CIs), cortex endodermis initials (CEIs), and vascular initials. The abundance and variety of factors participating in the establishment and maintenance of these cell types and their abundant and tight interconnections make it difficult to decipher system-level rules governing the process. Thus, we finally emphasize how a systems-level perspective, encouraged and favored by recent technical and methodological advances, could help to unravel the complete picture of root development and, particularly, SCN maintenance/differentiation.