The pRb-E2F pathway is involved in mediating diverse cell fates, and oncogenic disruption of the pathway is regarded as a hallmark of cancer. Recent studies highlighted the pRb-E2F axis as a regulator of a large gene network which includes RNA splicing and transcription targets. Here, we have performed a deep genome-wide analysis of differentially expressed genes (DEGs) and alternatively spliced (AS) RNA targets which highlighted broadly non-overlapping networks of genes that are independently regulated by the pRb-E2F pathway. Individual pathway components, including E2F1, pRb, and PRMT5, either as single or combined knockouts, were found to influence DEG and AS networks but to different extents. An analysis of the E2F1 interactome revealed SRSF2 and HNRNPC as candidate proteins that were able to functionally assist E2F1 in mediating AS events. Moreover, E2F1 AS activity was evident as cells progress through the cell cycle and during the DNA damage response, and apparent in tumour models. Our results highlight gene networks where transcription and splicing are linked and coordinated by the pRb-E2F pathway, and further establish the widespread influence that pRb, E2F1, and PRMT5 have on regulating biological diversity through RNA splicing control.