Cotton fibers are unicellular trichomes that differentiate from ovule epidermal cells and serve as an excellent model for the study of cell differentiation and cell elongation. However, we lack comprehensive knowledge of the regulatory networks and interactions of transcription factors implicated in fiber development. Here, we reported the construction of a high-throughput CRISPR/Cas9 mutant library targeting 662 cotton transcription factors preferentially expressed in fiber initiation and early elongation. In total, 2106 independent T0 plants were obtained. After low-cost barcode-based deep sequencing, the efficiency of gene editing was as high as 87.80%. Apparent morphological differences from the wild-type in terms of fiber traits were observed among 100 gene knockout mutants in a 3-year field trial. Compared with the wild type, most of the mutants exhibited shorter fiber and lower lint percentage. However, some negative regulatory factors regulating fiber length and lint percentage have also been identified. In addition, six genes related to fiber initiation, MYB25-like, MYB25, HD1, PDF2, MYB106 and HDG2, were characterized. Of these, overexpression of MYB25Like and HDG2 increased the number of fiber initials. Furthermore, a spatial-temporal protein–protein interactions network map was constructed using the high-density matrix-based yeast Y2H assay. Then, utilizing both LUC experiments and mutant population RNA-seq, a relay race regulatory model of early fiber development was established, with a four-tiered regulatory relationship as a central module. Biochemical experiments reveal that the genes in the regulatory module interact with one another in a complex fashion. These results provide new insights into the mechanistic regulation of fiber development.