For the second day in a row, the mayor presented the honor during his coronavirus briefing.
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First lady Chirlane McCray introduced the 64-year-old Lee and declared "today is a good day" despite the surge in COVID cases.
The seriousness of the situation was not lost on Spike, who quickly shifted to discussing the pandemic while accepting the honor.
"Driving here I heard about all these Broadway shows closing. We really can't get back to our true 'king of culture' until we rid of this virus because if artists can't perform in front of New Yorkers or the people who come from out of town. Culture is our DNA," Lee said.
The filmmaker has produced more than 35 films over the years.
Although not a native New Yorker, Lee grew up and spent much of his adult life in the city.
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"I love New York City," Lee said. "I'm a product of a New York City public education and it shaped me. I live and die New York City."
Feminist journalist and activist Gloria Steinem, 87, received the key to the city on Monday.
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