The vast amount of information contained in the genome is organized and packaged by chromatin into a cell’s nucleus. This chromatin is composed of protein complexes called nucleosomes, made up of histone proteins that can regulate accessibility and functionality of the underlying DNA. Canonical histones are synthesized and deposited during S-phase in a replication-coupled manner, meeting the demand for a massive number of new histones to package the newly synthesized DNA. There are also multiple variant histones, and these variants are thought to have different impacts on the DNA they bind. These variants can be deposited DNA synthesis-independently and provide replacement histones in terminally post-mitotic cells, including neurons. Encoded by two separates genes H3f3a and H3f3b, H3.3 is expressed throughout the cell cycle, and incorporates preferentially at enhancers, promoters, and gene bodies, suggestive of a function in gene regulation. H3.3 is known to play a role in developmental processes, including neural crest cell differentiation, gametogenesis, and zygotic genome activation. In the brain, silencing of H3.3 genes by RNA interference leads to deficits in neuronal layer distribution, activity- and environmental enrichment-induced gene expression, and memory Here, I leverage stage-dependent deletion of H3.3 genes from: 1) cycling neural progenitor cells, 2) neurons immediately after terminal mitosis, or 3) several days later, revealing the first post-mitotic days to be a critical window for de novo H3.3. Despite ubiquitous expression of H3.3 throughout cell types and the cell cycle, newborn cortical neurons undergo substantial de novo accumulation of H3.3 after becoming terminally post-mitotic. Deletion of H3.3 prior to this critical window abrogated this accumulation and had a profound impact on the establishment of the neuronal transcriptome. H3.3 is known to play a role in cell state transition, and the genes found to be most affected were typically upregulated across development, and often associated with a bivalent or “poised” promoter in neural progenitors. Coincident with these transcriptomic changes, I observed severe deficits in the formation of cortical axon tracts, including agenesis and misrouting of key intracortical connections and complete loss of the cortical spinal tract. Neuronal identity was also affected, and the typical laminar identity of deep-layer projection neurons failed to undergo normal refinement, resulting in mixed identities. After H3.3 accumulation within this developmental window, co-deletion of H3f3a and H3f3b caused progressive loss of H3.3 over several months without significant disruption of the transcriptome. The loss of H3.3 was not accompanied by an observable decrease in overall H3 levels or in H3 PTMs. Through this work I uncovered the potential existence of a non-H3.3 source of H3 that can compensate for the loss of H3.3 once the neuronal transcriptome is established, but not before. My work uncovers an active role of H3.3 in establishing transcriptional landscape and molecular identity in developing neurons immediately post-mitosis that is distinct from its role in maintaining histone H3 levels over the neuronal lifespan. These findings lead to further questions on the importance of H3.3 vs. histone turnover, and has important implications for histone variant regulation of developmental processes.