Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to investigatethe effect of the Pbx1 gene on the gene expression program of mature uterine NK cells. Methods: The differential analysis of gene expression profiles in uterine NK cells from Pbx1f/f and Pbx1f/f; Ncr1Cre mice at gd11.5 by RNA-Seq . Results: Using an optimized data analysis workflow, we found that the inactivation of PBX1 altered the NK cell gene expression program, and many gene expression changes, including significant down-regulation of Ptn and Ogn expression. Conclusions: PBX1 not only regulates the proliferative capacity of NK cells but also promotes the expression of GPFs in NK cells. Overall design: Uterine NK cells mRNA profiles of Pbx1f/f and Pbx1f/f; Ncr1Cre mice at gd11.5 were generated by deep sequencing, in duplicate, using Illumina HiSeq X Ten.