Platelet dense granules (DGs) are storage organelles for calcium ions, small organic molecules such as ADP and serotonin, and larger polyphosphates that are secreted upon platelet stimulation to enhance platelet activation, adhesion, and stabilization at sites of vascular damage. However, the precise timing of DG formation and maturation has not been definitively characterized, and how and when DG membrane contents are delivered is not at all known. In this thesis, I will discuss two important findings relevant to DG biology. The first finding addresses the timing of maturation of DGs within differentiation of the platelet precursor cell, the megakaryocyte (MK). DGs are thought to fully mature within MKs prior to platelet formation. Here I challenge this notion by exploiting vital fluorescent dyes to distinguish mildly acidic DGs from highly acidic compartments in platelets and MKs. In mouse platelets, compartments labeled by mepacrine — a fluorescent weak base that accumulates in DGs — are readily distinguishable from highly acidic compartments, likely lysosomes that are labeled by the acidic pH indicator, LysoTracker, and from endolysosomes and alpha (α) granules. By contrast, in primary MKs and megakaryocytoid cell lines, MEG-01 and differentiated G1ME2, labeling by mepacrine overlapped nearly completely with labeling by LysoTracker. Fluorescent puncta that labeled uniquely for mepacrine were first evident in G1ME2-derived proplatelets, suggesting that DGs undergo a maturation step that initiates in the final stages of MK differentiation. The second finding addresses the putative DG transmembrane protein vesicular monoamine transporter 2 (VMAT2) and its localization throughout MK differentiation. VMAT2 has been implicated as the transporter responsible for serotonin uptake into DGs and is therefore a likely candidate for a DG protein. Within MKs, VMAT2 localizes to acidic endolysosomal compartments and partially to CD63-containing structures. VMAT2 does not localize to early endosomes. However, as G1ME2 MKs differentiate to proplatelets, VMAT2 cosegregates with mepacrine from acidic endolysosomes, likely multivesicular bodies, suggesting that DG membrane sorting occurs during proplatelet differentiation. VMAT2 does not bind to adaptor protein (AP) complexes, but may employ an acidic cluster to effect localization. Together, these two studies provide insight into the timing and maturation of DG biogenesis during MK differentiation.