Each cotton fiber is a single cell trichome structure on cotton ovule epidermis. To improve the cotton fiber quality and product, we have to learn how the cell fate is determined. Transcriptome analyses were performed to compare genes expression changes in laser capture microdissected fiber cell initials (or epidermis) and inner ovules. Gene ontology and clustering analysis of differentially expressed genes indicated dynamic changes and display the potential genes determine the cotton fiber cell fate. A gene encoding transcription factor MYB2 and its downstream gene RDL1 were detected actively expressed at cotton fiber initiation stage. The transgenic plants of Arabidopsis showed that GFP:GhRDL1 and GhMYB2:YFP were co-localized in the nuclei of ectopic trichomes in siliques. Overexpressing GhRDL1 or GhMYB2 in A. thaliana Columbia-0 (Col-0) activated fiber-like hair production in 4-6% of seeds and had on obvious effects on trichome development in leaves or siliques. Co-overexpressing GhRDL1 and GhMYB2 in A. thaliana Col-0 plants increased hair formation in ∼8% of seeds. Overexpressing both GhRDL1 and GhMYB2 in A. thaliana Col-0 try mutant plants produced seed hair in ∼10% of seeds as well as dense trichomes inside and outside siliques, suggesting synergistic effects of GhRDL1 and GhMYB2 on fiber(trichome) cell fate determination. These data suggest that a different combination of factors is required for the full development of trichomes (hairs). A. thaliana can be developed as a model a system for discovering additional genes that control seed hair development in general and cotton fiber in particular.