We have identified a parallel G-quadruplex (R1WT) in the distal promoter region (-821 base-pairs upstream of the TSS) of the pluripotent gene REX1. Through biophysical and biochemical approach, we have characterized the G-quadruplex (GQ) as a potential molecular switch that may control REX1 promoter activity to determine the transcriptional fate. Small- molecule interactive study of the monomeric form of R1WT (characterized as R1mut2) with TMPyP4 and BRACO-19 revealed GQ destabilization upon interaction with TMPyP4 and stabilization upon interaction with BRACO-19. This distinctive drug interactivity suggests the in cellulo R1WT to be a promising drug target. The endogenous existence of R1WT was confirmed by BG4 antibody derived chromatin immunoprecipitation experiment. Here in, we also report the endogenous interaction of GQ specific transcription factors (TFs) with R1WT region in the human chromatin of cancer cell. The wild-type G-quadruplex was found to interact with four important transcription factors, (i) specificity protein (Sp1) (ii) non-metastatic cell 2 (NM23-H2): a diphosphatase (iii) cellular nucleic acid binding protein (CNBP) and (iv) heterogenous nuclear ribonucleoprotein K (hnRNPK) in the REX1 promoter. In contrast, nucleolin protein (NCL) binding was found to be low to the said G-quadruplex. The flexibility of R1WT between folded and unfolded states, obtained from experimental and computational analysis strongly suggests R1WT to be an important gene regulatory element in the genome. It controls promoter DNA relaxation with the coordinated interaction of transcription factors, the deregulation of which seeds stemness characteristic in cancer cells for further metastatic progression.