β-Cell regeneration is a key goal of diabetes research. Progression through the cell cycle is associated with retinoblastoma protein (pRb) inactivation via sequential phosphorylation by the “early” cyclins and cyclin-dependent kinases (cdks) (d-cyclins cdk4/6) and the “late” cyclins and cdks (cyclin A/E and cdk1/2). In β-cells, activation of either early or late G1/S cyclins and/or cdks is an efficient approach to induce cycle entry, but it is unknown whether the combined expression of early and late cyclins and cdks might have synergistic or additive effects. Thus, we explored whether a combination of both early and late cyclins and cdks might more effectively drive human β-cell cell cycle entry than either group alone. We also sought to determine whether authentic replication with the expansion of adult human β-cells could be demonstrated. Late cyclins and cdks do not traffic in response to the induction of replication by early cyclins and cdks in human β-cells but are capable of nuclear translocation when overexpressed. Early plus late cyclins and cdks, acting via pRb phosphorylation on distinct residues, complementarily induce greater proliferation in human β-cells than either group alone. Importantly, the combination of early and late cyclins and cdks clearly increased human β-cell numbers in vitro. These findings provide additional insight into human β-cell expansion. They also provide a novel tool for assessing β-cell expansion in vitro.