ABSTRACT Unicellular Leishmania parasites cause leishmaniases and require a life cycle stage-specific surface coat for pathogenesis. A major protein component of this coat is δ-amastins, which are human infective amastigote life cycle stage-specific transmembrane glycoproteins. Leishmania genes are encoded in co-transcribed gene arrays with little gene-specific transcriptional control. Here, we show that the Leishmania mexicana ortholog of ESB1 (denoted LmxESB1), a nuclear protein we previously identified in the related parasite Trypanosoma brucei, is required for δ-amastin regulation. LmxESB1 localises to a promastigote-specific nuclear body and its deletion caused derepression of δ-amastin expression from specific chromosomal loci. This upregulated δ-amastin phenotype was unstable, recovering over time. Transcriptomic analysis of recovered mutants identified two additional factors – LmxM.34.0190, an NIF-like phosphatase NIFP1, and LmxM.23.0730, the RNA-binding protein RBP10 – where deletion of which also resulted in δ-amastin misexpression. We have therefore identified a novel Leishmania nuclear protein that contributes to δ-amastin repression in promastigotes and factors that act in parallel with LmxESB1. This expands our understanding of how Leishmania controls stage-specific gene expression of surface coat proteins necessary for pathogenicity.