Emerging evidence reveals the crucial role of ubiquitous splicing factors in neural crest (NC) development, which is highly susceptible to splicing dysregulation. How these factors integrate with the NC gene regulatory network (GRN) and contribute to the increased vulnerability of NC to splicing perturbations are not well understood. Here, we show that NC-specific DLC1, in association with the SF3B1-PHF5A splicing complex, are crucial for determining avian trunk NC cell fate by regulating the splicing of pre-mRNA for NC specifiers SOX9 and SNAI2 rather than their upstream regulators BMP4, WNT1, and PAX7 in the GRN. Mechanistically, SF3B1-PHF5A are associated with the branch point adenosine (BPA) of all the factors’ introns, while DLC1 binds specifically to the BPA at the introns of SOX9 and SNAI2, thereby determining the functional specificity of the SF3B1-PHF5A complex in NC specification. Moreover, DLC1 enhances the susceptibility of NC cells to the splicing modulator pladienolide B by reducing the binding capacity of the SF3B1-PHF5A splicing complex to the shorter length of both SOX9 intron 2 and SNAI2 intron 1, which have weaker polypyrimidine tract 3’ of the BPA, resulting in intron retention and loss of NC progenitors. Our data highlight the critical role of the DLC1-SF3B1-PHF5A splicing complex in determining trunk NC cell fate and conferring its increased susceptibility to splicing perturbation.