A phosphoinositide signalling cycle is present in the nucleus, independent of that which occurs at the plasma membrane. The key enzyme involved in this cycle is phospholipase (PLC) β1. This nuclear cycle has been shown to be involved in both cell proliferation and differentiation. Here, we report that nuclear PLCβ1 activity is upregulated during differentiation of 3T3-L1 adipocytes. During differentiation there are two phases of PLCβ1 activity; the first occurs within 5 min of treatment with differentiation media, does not require new PLCβ1 to enter the nucleus and is regulated by pERK and PKC α while the second phase occurs from day 2 of differentiation, requires new PLCβ1 protein to enter the nucleus and is independent of regulation by pERK and PKC α. Over-expression with the PLC mutants, Δmk (which lacks the ERK phosphorylation site) and M2B (which lacks the nuclear localisation sequence), revealed that both phases of PLCβ1 activity are required for terminal differentiation to occur. Inhibition of PLCβ1 activity prevents the upregulation of cyclinD3 and cdk4 protein, suggesting that PLCβ1 plays a role in the control of the cell cycle during differentiation. These results indicate nuclear PLCβ1 as a key regulator of adipocyte differentiation.