The periodontal ligament (PDL) is a key contributor to the process of regeneration of the periodontium. The heterogeneous nature of the PDL tissue, its development during early adulthood, and the different conditions to which the PDL tissue is exposed to in vivo impart on the PDL unique characteristics that may be of consequence during its cultivation in vitro. Several factors affecting the in vivo setting influence the behaviour of PDL fibroblasts in culture. The purpose of this review is to address distinct factors that influence the behaviour of PDL fibroblasts in culture -in vivo-in vitro transitions, cell identification/isolation markers, primary PDL cultures and cell lines, tooth-specific factors, and donor-specific factors. Based on the reviewed studies, the authors recommendations include the use of several identification markers to confirm cell identity, use of primary cultures at early passage to maintain unique PDL heterogeneic characteristics, and noting donor conditions such as age, systemic health status, and tooth health status. Continued efforts will expand our understanding of the in vitro and in vivo behaviour of cells, with the goal of orchestrating optimal periodontal regeneration. This understanding will lead to improved evidence-based rationales for more individualized and predictable periodontal regenerative therapies.