Although major splicing regulatory mechanisms rely on the presence of cis-acting sequence elements in the precursor messenger RNA (pre-mRNA) to which specific protein and factors bind, splice choices are also influenced by transcription kinetics, promoter-dependent loading of RNA-binding proteins and nucleo-cytoplasmic distribution of splicing regulators. Within the highly crowded eukaryotic nucleus, molecular machines required for gene expression create specialized microenvironments that favor some interactions while repressing others. Genes located far apart in a chromosome or even in different chromosomes come together in the nucleus for coordinated transcription and splicing. Emerging tools to dissect gene expression pathways in living cells promise to provide more detailed insight as to how spatial confinement contributes to splicing control.