The epigenome bestows an interpretive layer to the genome that provides instructions as to which genes may be transcribed by the cell. As such, epigenetic modifications underpin the critical first cell lineage separation in the early embryo and orchestrate further differentiation of cells throughout development. While certain epigenetic modifications such as histone methylation are better-understood, other modifications such as histone citrullination and its conferring enzyme family, the peptidylarginine deiminases (PADIs), are relatively unexplored, particularly in a developmental context. Recent work has illuminated the importance of the PADI4 protein in the first cell fate decision and the maintenance of embryonic stem cell (ESC) pluripotency. However, the role of the PADIs and histone citrullination in trophoblast stem cells (TSCs) remains unknown. TSCs are the precursor cells of the major placental structures and represent the extraembryonic counterpart to ESCs. As such, the focus of this project was to produce the first in-depth characterization of the expression and function of the Padi genes and their epigenetic mark histone citrulline in murine TSCs. In this thesis, I report on several novel findings, including the expression of Padi2, Padi3, and Padi4 in TSCs, which is highest in the stem cell condition and decreases upon differentiation. Additionally, I identify the accumulation of histone H3 citrulline at chromocenters and its association with other epigenetic repressive marks, such as the KAP1 protein and H3K9me3. I also present the first Padi gene knockout (KO) study in TSCs with the generation of Padi2, Padi3, and Padi4 KOs and a Padi2/3 double KO. While the phenotypic analysis of these KOs revealed subtle changes like decreased proliferation and increased nuclear size, a readout of differentiation in trophoblast cells, none of the KOs cause an overt exit from the stem cell state. Notably, gene expression analysis via RNA-seq illustrated an apparent loss of heterogeneity amongst KO clones compared to the wild-type (WT), indicating a reduction of the plasticity required of multipotent stem cells in these KOs. Moreover, individual KOs had distinct effects on transcriptional profiles, highlighting the unique, non-redundant roles of the PADI proteins in TSCs. Finally, my work illustrates a nuclear role for PADI3, which has previously not been reported. I also identify Padi3 as a potential TSC marker gene which may possess a role in the earliest lineage separation in the blastocyst. Altogether this work provides new insights into the function of the PADIs and histone citrullination in a developmental context.